ISEV2019 Abstract Book * (BUTTON) Article notes * (BUTTON) Copyright and License information Collection date 2019. © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ([19]http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. [20]PMC Copyright notice PMCID: PMC6493311 __________________________________________________________________ About ISEV The International Society for Extracellular Vesicles is the leading professional society for researchers and scientists involved in the study of microvesicles and exosomes. With nearly 1000 members, ISEV continues to be the leader in advancing the study of extracellular vesicles. Founded in 2012 in Sweden, ISEV has since moved its headquarters to the USA. Through its programmes and services, ISEV provides essential training and research opportunities for those involved in exosome and microvesicle research. Mission statement Advancing extracellular vesicle research globally. Vision Our vision is to be the leading advocate and guide of extracellular vesicle research and to advance the understanding of extracellular vesicle biology. ISEV2019 Annual Meeting The International Society for Extracellular Vesicles is the premier international conference of extracellular vesicle research, covering the latest in exosomes, microvesicles and more. With an anticipated 1000 attendees, ISEV2019 will feature presentations from the top researchers in the field, as well as providing opportunities for talks from students and early career researchers. ISEV2019 International Organizing Committee IOC Chair: Hidetoshi Tahara, Ph.D. (Japan); Andrew Hill, Ph.D. (Australia), Carolina Soekmadji, Ph.D. (Australia), Cherie Blenkiron, Ph.D. (New Zealand), Edit Buzas, Ph.D. (Hungary), Hang Hubert Yin, Ph.D. (China), Juan Falcon Perez, Ph.D. (Spain), Kazunari Akiyoshi, Ph.D. (Japan), Kenneth W. Witwer, Ph.D. (USA), Kyoko Hida, Ph.D. (Japan), Lei Zheng (China), Marca Wauben, Ph.D. (The Netherlands), Mariko Ikuo, Ph.D. (Japan), Masahiko Kuroda, M.D., Ph.D. (Japan), Nobuyoshi Kosaka, Ph.D. (Japan), Ryou-u Takahashi, Ph.D. (Japan), Sai-Kiang Lim, Ph.D. (Singapore), Susmita Sahoo, Ph.D. (USA), Takahiro Ochiya, Ph.D. (Japan), Tang-Long Shen, Ph.D. (Taiwan), Yong Song Gho, Ph.D. (Korea) and Yoshinobu Takakura, Ph.D. (Japan) Journal of extracellular vesicles: Editors-in-Chief Clotilde Thery, Ph.D. (France) graphic file with name ZJEV_A_1593587_UF0001_C.jpg [21]Open in a new tab Plenary Session 1: Standardizations Chairs: Andrew Hill; Hidetoshi TaharaLocation: Level 3, Hall B NanoCosmos: extracellular vesicles as nanosized extracellular organelles delivering the complex messages between cells and organisms Yong Song Gho Department of Life Sciences, POSTECH, Pohang, Republic of Korea The secretion of nanosized lipid bilayered extracellular vesicles is a universal cellular process occurring from simple organisms to complex multicellular organisms. Recent progress in this area has revealed that extracellular vesicles play multifaceted pathophysiological functions by delivering the complex messages between cells and organisms, suggesting that extracellular vesicles are NanoCosmos, i.e., extracellular organelles that play diverse roles in intercellular and interkingdom communication. This presentation briefly introduces our last 20 year’s comprehensive research on extracellular vesicles derived from host, bacteria, diet and environments including their physical, biochemical and biological complex properties (http://evpedia.info). Then, this presentation focuses on our recent progress in novel extracellular vesicle-mimetic technologies for targeted drug delivery, theranostics and epigenetic reprogramming as well as for adjuvant-free, non-toxic vaccine delivery system against bacterial infection. Furthermore, bacterial extracellular vesicle-based cancer immunotherapy will be introduced. Based on the concept of emergent properties of heterogeneous extracellular vesicles, future research directions to decode the complexity of extracellular vesicle-mediated intercellular communication network, either at the single vesicle level or at a systems level as a whole, and the secret of life will be briefly introduced. Symposium Session 1: Cardiovascular Disease Thursday 25 April 2019 Chairs: J. Brian Byrd; Pia SiljanderLocation: Level B1, Hall B 11:00–12:30 OT01.01 Extracellular vesicles mediate neutrophil cell deployment from the spleen following acute myocardial infarction Naveed Akbar and Robin Choudhury University of Oxford, Oxford, UK Introduction: Acute myocardial infarction (AMI) mobilizes monocytes from the splenic reserve and induces transcriptional activation en route to the injured myocardium, possibly through interactions involving plasma liberated extracellular vesicles (EVs). Neutrophils also reside in the spleen and are the first cells to arrive at sites of injury and mediate further damage. Here, we describe neutrophil deployment from the spleen in AMI and by endothelial cell (EC)-derived EVs. Methods: Patients provided informed consent as part of the Oxford Acute Myocardial Infarction Study. EV were isolated using ultra centrifugation (120,000g 2 h) and characterized for size and concentration by Nanoparticle Tracking Analysis, EV markers (TSG101, ALIX, CD63/CD69) by western blot, and microRNAs (miRNAs) by RT-qPCR. Mouse and human EC were used in vitro to derive EC-EV. Results: Patients presenting with AMI (n = 15) have 2.2-fold more plasma EV at time of injury vs. a 6-month follow-up measurement (P = 0.008). Plasma EVs at the time of presentation correlate significantly with the extent of ischemic injury (R = 0.046, P = 0.006) and plasma neutrophils (R = 0.37, P = 0.017). Experimental AMI in wild type, naïve (C57B6/J) mice induces splenic-neutrophil deployment (P = 0.004). Human plasma EV-miRNAs are significantly altered post-AMI. AMI plasma EV-miRNA-mRNA targets (IPA, Qiagen) are significantly over represented when compared to neutrophil Gene Ontology terms for degranulation (P < 0.001), activation (P < 0.001), chemotaxis (P = 0.008) and migration (P = 0.008). Human EC releases more EV after inflammatory stimulation (control 2.4 × 108 ± 4.9 x 107 EVs/mL vs. tumour necrosis factor-alpha stimulated, 1.4 × 109 ± 3.0 × 108 EVs/mL, P = 0.003) and contains many of the miRNAs enriched in human plasma-EV following AMI. Mouse EC-EV tail vein injected into otherwise wild-type, naïve mice mobilize splenic neutrophils to peripheral blood (P < 0.001). Summary/Conclusion: Neutrophils appear at sites of injury in the immediate hours after ischemic injury. Neutrophil interactions with EC-EV may mediate their splenic liberation and transcriptional programming following AMI, en route to the injured myocardium. The splenic neutrophil reserve may be a novel therapeutic target in AMI. Funding: British Heart Foundation. OT01.02 In vivo characterization of endogenous cardiovascular extracellular vesicles and their response to ischaemic injury Aaron Scott ^a, Costanza Emanueli^b and Rebecca Richardson^c ^aUniversity of Bristol, Uffculme, UK; ^bImperial College London, London, UK; ^cUniversity of Bristol, Bristol, UK Introduction: Cardiomyocytes and endothelial cells are counted among the cell types that secrete extracellular vesicles (EVs). EVs mediate the targeted transfer of lipids, proteins and nucleic acids by traversing the extracellular milieu. Recent studies suggest that EVs play a functional role in cardiovascular disease and cardiac repair. For example, a population of exosomes carrying proangiogenic miRNAs was found in the pericardial fluid of patients undergoing heart surgery. Further investigation will be required to determine which cardiac cells are producing these EVs, the cell type receiving them and the functional relevance of this. Methods: A complete understanding of this process requires a comprehensive in vivo model. The zebrafish is an amenable vertebrate model with genetic tractability and optical transparency allowing for subcellular observation in a living organism. The use of stable transgenic lines with cell-type-specific promoters driving the expression of membrane tethered fluorophores allows labelling of the cell membrane and the EVs produced by individual cell types. Light sheet microscopy permits cardiovascular-specific EVs to be tracked in vivo and an established ischaemic injury model allows EV profiles from uninjured, injured and repairing/regenerating cardiac tissue to be determined and compared. Results: Live imaging of transgenic zebrafish with endothelial cell-derived EVs labelled with mCherry reveals large numbers of EVs in the peripheral circulation, interactions with downstream endothelial cells and release in to the blood flow from filopodia-like protrusions. Cardiomyocyte-derived EVs are observed in the pericardial fluid surrounding the heart and are often seen interacting with cells of the pericardial wall. Additionally, a modified FACS protocol reveals how cardiomyocyte-derived EV numbers fluctuate in response to cardiac injury. Summary/Conclusion: This data present exciting opportunities to further dissect the cargo being carried by these EVs in a vertebrate model of human disease. Funding: British Heart Foundation. OT01.03 Enhanced fibrinolysis and altered extracellular vesicles after remote ischaemic preconditioning in non-diabetic coronary artery disease patients Caroline J. Reddel ^a, Jerrett Lau^b, Gabrielle Penning^c, Vivien Chen^d and Leonard Kritharides^e ^aANZAC Research Institute, University of Sydney, Concord Repatriation General Hospital, Concord, Australia; ^bDepartment of Cardiology, Concord Repatriation General Hospital, Concord, Australia; ^cANZAC Research Institute, University of Sydney, Concord Repatriation General Hospital, Concord, Australia; ^dANZAC Research Institute and Department of Haematology, Concord Repatriation General Hospital, Concord, Australia; ^eANZAC Research Institute and Department of Cardiology, Concord Repatriation General Hospital, Concord, Australia Introduction: Brief non-harmful ischaemia, remote ischaemic preconditioning (RIPC) has been shown to confer benefit to patients with coronary artery disease (CAD). Some studies indicate lesser benefit in patients with diabetes. RIPC may enhance fibrinolysis. Hypothesis: RIPC causes an increase in fibrinolytic potential through release of fibrinolytic factors from the endothelium or fibrinolysis-supporting extracellular vesicles (EVs) and this effect is less evident in patients with diabetes. Methods: Patients at Concord Hospital with suspected CAD gave written informed consent and were administered RIPC (sphygmomanometer on the arm, 3 × 5 min cycles, n = 31) or sham (n = 29) before angiography, with recruitment ongoing. Blood was collected pre- and immediately post-RIPC/sham and platelet-free plasma generated. Global coagulation/fibrinolytic potential was measured by overall haemostatic potential assay (Reddel et al. Thromb Res. 2013; 131(5): 457–462) and various fibrinolytic factors by ELISA. EV were assessed by flow cytometry (Reddel et al. Thromb Haemost. 2018; 118(4): 723–733) using fluorescent surface markers for phosphatidylserine and cell origin including platelets (CD41a), leukocytes (CD45) and MAC-1 (CD11b). Positive events were defined with supernatant of ultracentrifuged pooled normal plasma as negative control. Changes pre–post RIPC were assessed by paired t-test. The study was approved by the local ethics committee. Results: In the whole population, there was no effect of RIPC on fibrinolytic factors but a decrease in platelet-derived EV. However, in non-diabetic patients and not in diabetic patients, RIPC increased overall fibrinolytic potential and CD45+ and CD11b+ EV. These effects were not seen after sham treatment. Summary/Conclusion: There is a global increase in fibrinolytic potential after RIPC treatment in CAD patients without diabetes mellitus, which may be contributed to by increased leukocyte-derived EV and/or decreased platelet-derived EV. Ongoing work aims to directly identify this contribution in patients who undergo RIPC. OTO1.04 Urinary extracellular vesicle concentration, microRNA-155 expression and inflammatory surface marker expression are altered in patients with symptomatic coronary artery disease Stephen Fitzsimons ^a, Silvia Oggero^b, Niall Mahon^c, Nicola Ryan^c, Mauro Perretti^d and Orina Belton^a ^aUniversity College Dublin, Dublin, Ireland; ^bQueen Mary University of London, London, UK; ^cThe Mater Misericordiae University Hospital, Dublin, Ireland; ^dWilliam Harvey Research institute, Queen Mary University of London, London, UK Introduction: Urinary extracellular vesicles (uEVs) (exosomes, microvesicles and apoptotic bodies) have potential as diagnostic and prognostic biomarkers. In atherosclerosis, the underlying cause of heart attack and stroke, EV release can be dysregulated and their contents can mediate pro-inflammatory effects. Several markers have been previously identified on uEV including exosome markers CD63 and CD9, CD45 (leukocyte marker), CD61 (platelet marker), CD14 (monocyte/macrophage marker) and α/β integrins. The selectively packaged cargo of these membrane bound carriers include microRNAs (miRs). miR-21 and miR-155 are key regulatory miRs that are upregulated in immune cells and are released in EVs following exposure to pro-inflammatory stimuli. miR-155 has been reported to have pro-atherogenic effects and miR-155 deficiency in murine models results in reduced atherosclerotic lesion burden. Methods: Urine was collected from patients diagnosed with coronary artery disease (CAD), classified as symptomatic (non-ST-elevation myocardial infarction, ST-elevation myocardial infarction or unstable angina) or asymptomatic (stable angina). uEVs from symptomatic and asymptomatic patients were isolated via benchtop centrifugation. The concentration and size of uEVs were analysed via the NanoSight NS300 (n = 15 per group). The expression of miR-155 and miR-21 was investigated by RT-qPCR (n = 10 per group). uEV surface marker expression was analysed by ImageStreamX MK2 Imaging Flow Cytometer (12 per group). Results: uEV concentration in symptomatic patients (median; 6.46E+9 particles/mL) was significantly decreased (p < 0.05) compared to asymptomatic patients (median; 1.25E+10 particles/mL). CD11B+ uEVs were increased and CD16+ uEVs were decreased in the symptomatic patients (p < 0.01). In addition, the concentration of CD45+ EVs were increased in symptomatic patients (p < 0.001). Although uEV miR-21 was unchanged, miR-155 expression was significantly increased in the symptomatic group (p < 0.05). Summary/Conclusion: uEV concentration, miR-155 expression and surface marker expression have diagnostic and prognostic potential. As CAD severity increases, uEV concentration is reduced, surface marker expression is altered and uEV miR-155 expression is increased. Funding: The Irish Research Council. OT01.05 Circulating extracellular vesicle-associated microRNAs as predictive biomarkers of cardiovascular complications in end-stage renal disease Dakota D. Gustafson ^a, Jessica Fitzpatrick^b, Jason Fish^c and Rulan Parekh^b ^aDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; ^bChild Health Evaluative Sciences, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada; ^cToronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada Introduction: Chronic kidney disease affects roughly three million Canadians and is accompanied by considerable human suffering, premature death and costly medical care. The incidence of ESRD and its associated cardiovascular (CV) complications continues to rise despite improved treatments for the primary etiologies of diabetes and hypertension. Methods: We are using multiple biological approaches to better understand the organ-level effects of extracellular vesicle (EV)/miRNA dysregulation and gather the detailed mechanistic insight necessary for the development of an integrative CV risk prediction model for diabetic ESRD patients. The foundation of this study is the Predictors of Arrhythmic and Cardiovascular Risk in End Stage Renal Disease (PACE) cohort (n = 571); a cohort of ESRD patients followed over four years, with a repository of clinical data with matched biological samples. ExoQuick was used for the isolation of EVs from plasma, and their properties characterized through NanoSight analysis, western blotting and electron microscopy. A high-throughput microfluidics RT-qPCR platform (n = 420) was used to examine potential associations between miRNA and clinical outcomes. We then validated functionality on human coronary endothelial cells (n = 12, coronary artery disease vs. control) using mRNA-Seq. Results: EV analysis revealed diabetic ESRD patients have increased circulating vesicle concentrations; in addition to having vesicles of a larger mean size. Data from our clinically translatable microfluidics-based RT-qPCR methodology identified numerous potential microRNA biomarkers for CV complications of ESRD. Following miRNA identification, we utilized penalized regression models to generate a panel of miRNAs which may serve as CV-risk predictors for diabetic ESRD patients. In particular, miR-23b-3p appears to be significantly associated with coronary artery disease severity. Summary/Conclusion: This data will be weighted with the novel biomarker data and fully integrated to build a clinical risk prediction model for the development of CV complications in ESRD and reassessed in a new cohort (D4 Cohort) replicate the findings and validate the risk prediction model. Funding: This work was funded by AstraZeneca and the Canadian Vascular Network. OT01.06 Live tracking system for endogenous exosomes Weijia Luo^a, Yuan Dai^a, Kelsey Andrade^a, Megha Chandna^b, Pamela Ulloa-Franco^c and Jiang Chang ^a ^aTexas A&M University College of Medicine, Houston, USA; ^bUniversity of Texas-Austin, Houston, USA; ^cTrinity College, Hartford, USA Introduction: Exosomes are emerging new category of messengers that communicating among cells, tissues and organs. Understanding the kinetic of exosome communication in vivo is a critical foundation for studying exosome functions and developing exosome-based drug-delivery models. Current studies of exosome in vivo trafficking largely rely on the administration of exogenous exosomes labelled by fluorescent dyes or proteins. These methods may not fully represent endogenous exosome kinetics due to ex vivo exosome manipulation. Here, we established the first inducible endogenous exosome tracking mouse model that tracks endogenous exosome released by cardiomyocytes in vivo. Methods: We generated a transgenic mouse expressing the bioluminescent reporter Nano-luciferase (NanoLuc)-fusion protein. The ultrasensitive and stable NanoLuc reporter has a 150-fold stronger signal compared to the traditional Firefly and Renilla luciferases and the longest luminescent half-life amongst all known luciferases. We fused NanoLuc reporter with exosome surface marker CD63 for specific labelling of exosomes. Then, the cardiomyocyte-specific αMHC promoter followed by a loxP-STOP-loxP cassette was engineered for precise spatial labelling of exosomes originating from cardiomyocytes. We then crossed the cardiomyocyte-specific mouse with a tamoxifen-inducible Cre mouse (R26CreERT2) to achieve an inducible system. The exosome labelling and distribution were assessed by luciferase assay and noninvasive bioluminescent live imaging. Results: CD63NanoLuc expression was tightly controlled and only detected in cardiomyocytes upon induction. The endogenous exosomes released from cardiomyocytes were labelled and detected in vitro in cell culture supernatant, and in vivo in animal plasma. A signature distribution profile of the endogenous cardiomyocyte-releasing exosomes was achieved. Summary/Conclusion: This exosome tracking model enables elucidating the endogenous exosome trafficking pattern, and allows the study of exosome behaviour under different conditions. It will provide a powerful tool for the exploration of the biological functions, mechanisms and clinical applications of exosomes in a broad spectrum of research. Funding: AHA Innovative Project Award: 18IPA34180012. Symposium Session 2: Nucleic Acid Biomarkers in Human Disease Chairs: Robert Kitchen; Louise LaurentLocation: Level B1, Lecture Room 11:00–12:30 OT02.01 miRNA exosomal biomarkers in brain derived and serum exosomes associated with neurodegenerative diseases Lesley Cheng ^a, Xia Li^b, James Doecke^c, Laura Vella^d, Natasha Vassileff^e, Mitch C. Shambrook^a, Robyn Sharples^f, Saima Zafar^g, Inga Zerr^h, Catriona McLean^i, Malcolm Horne^d, Colin Masters^d, Kevin Barnham^d and Andrew Hill^j ^aDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, VIC, Australia, Melbourne, Australia; ^bDepartment of Mathematics and Statistics, La Trobe Institute for Molecular Science, La Trobe University, VIC, Australia, Melbourne, Australia; ^cCSIRO Digital Productivity Flagship/The Australian E-Health Research Centre, Herston, Queensland, Australia, Brisbane, Australia; ^dThe Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia; ^eThe Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia; ^fThe Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia; ^gDepartment of Neurology, Clinical Dementia Centre, University Hospital Göttingen, Göttingen, Germany; ^hThe Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia., Melbourne, Australia; ^iThe Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia Introduction: Several blood-based tests have been explored to detect Alzheimer’s disease (AD) and other neurogenerative diseases; however, evidence is required to determine whether blood sampling is an appropriate specimen to diagnose brain diseases. Exosomes are small extracellular membrane vesicles packaged with RNA and protein cargo. Previously we isolated serum exosomes from AD patients which displayed an abnormal composition of 16 specific microRNA (miRNA) biomarkers compared to controls. Methods: To provide evidence that our serum exosomal miRNA biomarkers are suitable for the detection of a brain condition, we also profiled exosomes isolated from post-mortem human AD (n = 8), PD (n = 8), ALS (n = 7) and control (n = 5–8 per group) brain tissues using next-generation sequencing. Results: Brain-derived exosomes (BDEs) were found to contain a unique profile of small RNA, including miRNA, compared to whole tissue. Furthermore, all 16 AD serum biomarkers, identified in our previous study, were detected in BDEs, together with differentiators for PD, ALS and CJD diagnosis in serum and in some cases neural-derived exosomes. Summary/Conclusion: This work has identified highly specific panels of miRNA that is both present in the brain and blood of AD, PD, ALS and CJD patients. The miRNA candidates can be used to develop a blood-based diagnostic test highly relevant to a brain disease, equivalent to noninvasive brain biopsy. Funding: National Health and Medical Research Council, Australia MND, Australia Creutzfeldt-Jakob disease Support Group Network, Australia Dementia Centre for Research Collaboration, Australia OT02.02 Brain-derived extracellular vesicle microRNA signatures associated with in utero and postnatal oxycodone exposure: Implications for altered synaptogenesis Victoria Schaal^a, Dalia Moore^b, Peng Xiao^a, Sowmya V. Yelamanchili^b, Gurudutt Pendyala ^a ^aUniversity of Nebraska Medical Center, Omaha, USA; ^bDepartment of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, USA Introduction: Oxycodone (oxy) is a semi-synthetic opioid commonly used as a pain medication which also is a widely abused prescription drug. While very limited studies have examined the effect of in utero oxy (IUO) exposure on neurodevelopment, a significant gap in knowledge is the effect of IUO compared with postnatal oxy (PNO) exposure on synaptogenesis – a key process in the formation of synapses during brain development in the exposed offspring. In the present study, we isolated and characterized brain-derived extracellular vesicle (BDE)-associated microRNA cargo from the brains of IUO and PNO offspring using RNA seq. Several key miRNAs unique to both the IUO and PNO groups were identified and validated using RT-PCR. To further gain mechanistic insights, we characterized the miRNA cargo effects on changes in synaptic architecture using in vitro primary neurons during a key stage of brain development. Methods: Density gradient EV isolations from brain tissue, transmission electron microscopy, RT-PCR, in vitro primary neuronal cultures and spine density analysis. Results: Transmission electron microscopy revealed an increase in BDE sizes in both the PNO and IUO groups suggesting that oxy exposure can affect BDE size thus indicating differential expression of molecular cargo. Next, RNA-Seq identified novel and distinct BDE miRNAs unique to IUO and PNO which were further validated by RT-PCR. Bioinformatics analysis on these differentially expressed BDEs, revealed key Gene Ontology terms involved in neurodevelopment such as neuron projection development, neuronal morphogenesis, pallium/cerebellum development in the IUO offspring. To determine, if BDEs impacted the synaptodendritic architecture, we treated 14 days in vitro rat cortical primary neurons with equal amounts of P14 BDEs from the three groups. Confocal imaging of dendritic spines showed a significant reduction on treatment with PNO BDEs and which was further exacerbated on treatment with the IUO BDEs. Summary/Conclusion: We conclude that BDEs from PNO and IUO offspring carry potentially distinct BDE miRNA cargo that subsequently damage the synaptodendritic architecture and could further lead to neuronal dysfunction at a key stage of neurodevelopment. Funding: Start-up funds and NIH/NIDA. OT02.03 Development of a high-performance urine exosomal-mRNA signature for identification of bladder cancer Sudipto Chakrabortty ^a, Robert Kitchen^a, James Hurley^a, Georg Stoll^b, Xuan Zhang^c, Mikkel Noerholm^d, Seth Yu^a and Johan Skog^e ^aExosome Diagnostics, Inc, Waltham, USA; ^bExosome Diagnostics, GmbH, Martinsried, Germany; ^cNeuology and Radiology Services and program in Neuroscience, Harvard Medical School, Massachusetts General Hospital, Boston, USA; ^dExosome Diagnostics, GmbH, Martinsried, Germany; ^eExosome Diagnostics, Inc., Waltham, Massachusetts, USA Introduction: Blood in the urine is a common symptom of bladder cancer but of individuals who present with haematuria on average only 8% will have cancer. Moreover, up to 70% of patients with a prior bladder tumour will experience a relapse. The majority of these individuals will therefore undergo invasive and expensive testing (cystoscopy & CT scan) to confirm the presence of a tumour, either for first diagnosis or active surveillance of recurrence. A low-cost, noninvasive urine test capable of preventing unnecessary biopsies is a challenging but attractive proposition. Methods: Here, we present results from a clinical study in which exosomal mRNAs were profiled from voided urine, collected prior to diagnosis, from individuals suspected of having either newly diagnosed or relapsed bladder cancer. We selected 81 individuals for the clinical study, 44 of whom were diagnosed with mostly early stage bladder cancer. The remaining individuals were healthy or diagnosed with a non-cancerous urinary disease. Results: We identified a 16-mRNA signature by mining over 25,000 public and proprietary RNA-seq datasets, using a machine learning approach to rank genes based on dysregulation in bladder cancer, presence in urine exosomes and stability to haematuria. Using this signature, we trained a classifier to differentiate samples based on presence/absence of bladder cancer, optimized for negative predictive value (NPV). The model performs well in both newly diagnosed and recurrent cases, even in low-grade disease, with an overall performance of 100% NPV at 46% specificity. As the model is based solely on exosomal mRNA abundance, the score provides entirely new information that would enable a clinician to further improve specificity by considering standard of care parameters. Summary/Conclusion: Exosomal mRNAs have been used to diagnose other malignancies but this represents the first application of this form of liquid biopsy to bladder cancer. While performance must be validated in a larger clinical trial, this signature could prevent ~50% of unnecessary biopsies, provide a noninvasive means of monitoring relapse and reduce the financial burden of early stage bladder cancer care. OT02.04 Genome-wide methylation profiling of extracellular vesicle DNA allows brain tumour classification Franz Lennard. Ricklefs ^a, Cecile Maire^b, Katharina Kolbe^b, Mareike Holz^b, Manfred Westphal^b, Ullrich Schüller^b and Katrin Lamszus^b ^aUniversity medical center Hamburg-Eppendorf, Hamburg, Germany; ^bUniversity Medical Center Hamburg-Eppendorf, Hamburg, Germany Introduction: Genome-wide methylation profiling has recently been developed into a tool that allows subtype tumour classification in central nervous system (CNS) tumours. Extracellular vesicles (EVs) are released by CNS tumour cells protecting their cargo, including DNA, from degradation rendering EVs as optimal biomarkers to define subgroups, stratify patients and monitor therapy by liquid biopsy. It is unclear, however, if DNA derived from glioma EVs reflects genome-wide methylation profiles and mutational statuses that would allow tumour classification. Methods: DNA was isolated from glioma cell cultures (GSC) EVs, GSCs and matched tumour samples (n = 3). EVs were isolated through differential ultracentrifugation and classified by nanoparticle tracking analysis (NTA), immunoblotting, imaging flow cytometry (IFCM), multiplex EV assay and electron microscopy. Genome-wide DNA methylation profiling was performed using a 850-k Illumina EPIC array and classified by the DKFZ brain tumour classifier. Results: GSCs secrete diverse EVs as measures by IFCM and multiplex EV assay that are high for common EV markers (a.e. CD9, CD63 and CD81). The range of EVs was 120–150 nm measured by NTA. Genome-wide methylation profiles of GSC EVs in addition to copy number alterations and mutations matched their parental GSC and original tumour sample, being Glioblastoma, IDH wildtype or mutant, with additional subclass analyses. Specifically, MGMT methylation statuses could be obtained through EV DNA. Summary/Conclusion: Here we report, that EV DNA reflects the tumour methylation class as well as most copy number variations and mutations present in the parental cells and the original tumour. DNA EV methylation profiles may therefore be used to detect and classify CNS tumours. Funding: FLR received a scholarship of the German Academic Foundation. OT02.05 Methamphetamine use disorder alters plasma extracellular vesicle characteristics and microRNA expression Ursula Sandau^a, John Nolan ^b, Xiao Shi^c, Tracy Swanson^c, Marilyn Huckans^d, William Schutzer^d, Kylie Sage^e, Jodi Lapidus^f, Jennifer Loftis^d, Aaron Janowsky^g and Julie A. Saugstad^a ^aDepartment of Anesthesiology & Perioperative Medicine, Oregon Health & Science University, Portland, USA; ^bScintillon Institute, San Diego, USA; ^cVA Portland Health Care System, and Department of Psychiatry, Oregon Health & Science University, Portland, USA; ^dVA Portland Health Care System, Department of Psychiatry, and Methamphetamine Abuse Research Center, Oregon Health & Science University, Portland, USA; ^eBiostatistics & Design Program, Oregon Health and Science University, Portland, USA; ^fBiostatistics & Design Program, Oregon Health and Science University, Oregon Health & Science University – Portland State University School of Public Health, Portland, USA; ^gVA Portland Health Care System, Departments of Psychiatry and Behavioral Neuroscience, and Methamphetamine Abuse Research Center, Oregon Health & Science University, Portland, USA Introduction: Methamphetamine’s (MA) rewarding properties and addictive potential are correlated with increased synaptic dopamine availability following alterations in dopamine and vesicular monoamine transporter function. We examined plasma extracellular vesicle (EV) number, size, protein markers and miRNA content in human subjects who are actively using MA. Methods: Plasma samples from 10 adults with active MA dependence (MA-ACT), and 10 non-dependent controls (CTL) were obtained from the Methamphetamine Abuse Research Center Biorepository at Oregon Health & Science University and the VA Portland Health Care System. We used single Vesicle Flow Cytometry to directly measure plasma EV concentration and size. We used size-exclusion chromatography (iZON Science) to isolate plasma EVs. EV total RNA isolated by mirVana™ PARIS™ RNA Kit (ThermoFisher) was analysed on TaqMan® Array Human MicroRNA A + B Cards Set v3.0 (ThermoFisher). MiRNA expression was compared between MA-ACT and CTL using two-sample t-tests for miRNA expressed in at least 50% of samples in at least one of the two groups. Tobacco use was controlled for. Results: The data show that in MA-ACT (n = 5) vs. CTL (n = 5), four of the five MA-ACT have an increase in total plasma EVs relative to all five CTL. The average EV concentration in MA users (2 × 108 ± 4.5 × 107 EV/μL) is trending to increased levels, relative to CTL (7.5 × 107 ± 0.9 × 107 EV/μL). EV counts relative to size show a range of EVs with a mode of ~110 nm in both MA-ACT and CTL plasma, and equivalent median EV size. Of 226 miRNA in the EVs, there are 30 miRNAs that meet have area under the curve (AUC) >0.65 and median difference >1, and 47 miRNAs with AUC >0.65 and mean difference >1. Twenty-three of these miRNAs overlap and are the current focus of target prediction. Summary/Conclusion: EV miRNA expression in subjects with MA use disorder was significantly different than in control participants, suggesting that MA may affect EV communication among cells. The differential miRNA expression also implicates a role for EVs in behavioural and physiological effects specific to MA and suggests that there may be changes in expression of miRNAs that are relevant to specific drugs of addiction, as well as to a spectrum of drug-mediated addiction disorders. OT02.06 Use of extracellular vesicles purified from lymphatic exudative seroma as surrogate markers of melanoma residual disease Susana Garcia-Silva ^a, Alberto Benito-Martín^b, Sara Sanchez-Redondo^c, Alberto hernandez-Barranco^a, Pilar Ximénez-Embún^d, Laura Nogués^a, Ana Isabel Amor^a, Kay Brinkmann^e, Marina S Mazariegos^c, Jasminka Boskovich^f, Mercedes Robledo^g, Johan Skog^h, Mikkel Noerholm^e, Javier Muñoz^i, Pablo L. Ortiz-Romero^j, José Luis Rodríguez-Peralto^k, Piotr Rutkowski^l and Héctor Peinado^a ^aMicroenvironment and Metastasis Laboratory, Molecular Oncology Programme. Spanish National Cancer Research Center (CNIO), 28029 Madrid, Spain, Madrid, USA; ^bChildren‘s Cancer and Blood Foundation Laboratories, Departments of Pediatrics, Weill Cornell Medicine, New York, NY 10021, USA, New york, USA; ^cMicroenvironment and Metastasis Laboratory, Molecular Oncology Programme. Spanish National Cancer Research Center (CNIO), 28029 Madrid, Spain, MAdrid, USA; ^dProteomics Unit – ProteoRed-ISCIII, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain, Madrid, USA; ^eExosome Diagnostics, GmbH, Martinsried, Germany, Martinsried, USA; ^fElectron Microscopy Unit, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain, MAdrid, USA; ^gHereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain, Madrid, USA; ^hExosome Diagnostics, Inc., Waltham, Massachusetts, USA, Waltham, USA; ^iSpanish National Cancer Research Centre, Madrid, South Georgia & South Sandwich Islands; ^jDepartment of Dermatology, Medical School, Universidad Complutense, Instituto i + 12, Hospital Universitario 12 de Octubre, Madrid 28041, Spain, Madrid, USA; ^kDepartment of Pathology, Medical School, Universidad Complutense, Instituto i + 12, Hospital Universitario 12 de Octubre, Madrid 28041, Spain, Madrid, USA; ^lMaria Sklodowska-Curie Institute – Oncology Center, Department of Soft Tissue/Bone Sarcoma and Melanoma, Warsaw, Poland, Varsaw, USA Introduction: Liquid biopsies in melanoma patients have the potential to improve prognosis. Exudative seroma obtained in the drainage implanted post-lymphadenectomy has never been explored as a source of biomarkers. The use of circulating extracellular vesicles (EVs) as surrogate markers of residual disease could be a novel and powerful non-invasive tool. Methods: Exosomes were purified by ultracentrifugation from exudative seroma obtained after lymphadenectomy from stage III melanoma patients, were analysed by nanosight analysis and electron microscopy, and compared to plasma in a total of 92 samples. We profiled the proteomic profiles of exudative seroma- and plasma-derived exosomes by mass spectrometry. Extracellular vesicle-associated nucleic acids (EV-NAs) and analysed BRAFV600E by an allele-specific PCR in exudative seroma samples as a novel parameter to detect residual disease Results: We found that exudative seroma is a novel biofluid highly enriched in EVs, higher size and increased DNA cargo in comparison to plasma. Proteomic analysis of seroma-derived exosomes demonstrated that they are enriched in melanoma oncogenic pathways together with immune-related pathways; however, proteomic analysis did not allow identify biomarkers of relapse or progression. Importantly, detection of BRAFV600E mutation in seroma-derived EVs obtained 24–48 hours post-lymphadenectomy identified patients at risk of relapse significantly (Log rank p = .0067) in a cohort of 17 stage III melanoma patients followed up for 700 days. Summary/conclusion: Our data show for the first time that exudative seroma obtained post-lymphadenectomy is a novel biofluid enriched on EVs and DNA that can be interrogated for melanoma markers and BRAF mutation. Analysis of BRAFV600 mutation identified patients at risk of relapse with high significance. These data support that our approach could be a novel factor to detect residual disease right after surgery in stage III melanoma patients. Our analysis could be a novel approach to aid oncologists to identify high-risk groups of patients post-lymphadenectomy and improve patient outcome. Funding: MINECO, NIH, Starr Foundation, Ramon y Cajal Programme, AECC, FERO foundation, and MINECO-REDiEX. Symposium Session 3: EVs in Cancer Metastasis and Angiogenesis Chairs: Kyoko Hida; Alissa WeaverLocation: Level 3, Hall B 11:00–12:30 OT03.01 Stem cell-derived extracellular vesicles increase cancer stem cell sensitivity to tyrosine kinase inhibitors through Akt/mTOR/PTEN-combined modulation Benedetta Bussolati ^a, Valentina Fonsato^b, Michela De Lena^c, Stefania Tritta^d, Alessia Brossa^b, Ruggero Calvetti^e, Ciro Tetta^f and Giovanni Camussi^g ^aDepartment of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy; ^bMolecular Biotechnology Center, University of Torino, Torino, Italy; ^cMolecular Biotechnology Center, University of Torino, Torino, Italy; ^dUniversità di Torino, Nichelino, Italy; ^eDepartment of Molecular Biotechnology and Health Sciences, Torino, Italy; ^fUnicyte srl, Turin, Italy; ^gDepartment of Medical Sciences, University of Turin, Turin, Italy Introduction: Cancer Stem Cells (CSCs) are a small cell population able to sustain the maintenance and recurrence of tumours. In consideration of the high drug resistance and tumour initiating capability, targeting CSCs represents an important approach to eradicate tumours. We previously showed a pro-apoptotic effect of extracellular vesicles (EVs) derived from human liver stem cells. In this study, we evaluated whether HLSC-EVs could act in synergy with tyrosine kinase inhibitor drugs (TKIs) on apoptosis of CSCs isolated from renal carcinomas. Methods: We administered HLSC-EVs and TKIs to renal CSCs, as co-incubation or sequential administration. TKIs were also loaded in EVs. Intracellular phosphoproteins were evaluated in the CSC lysates by the magnetic bead-based immunoassays Bio-Plex Pro cell-signalling assay and confirmed by Western Blot analysis. Results: We found that HLSC-EVs in combination with Sunitinb or Sorafenib significantly increased renal CSCs apoptosis induced by low TKI dose. At variance, no synergistic effect was observed when bone marrow mesenchymal stem cell-derived EVs were used. CSC apoptosis was also enhanced when TKIs were loaded in HLSC-EVs. In particular, renal CSCs chemosensitivity to TKIs was enhanced when HLSC-EVs were either co-administered with TKIs or added after, but not before. By a mechanistic point of view, Akt/mTOR and Erk and Creb intracellular pathways, known to be pivotal in the induction of tumour growth and survival, appeared modulated as consequence of TKIs/HLSC-EVs co-administration as well as by EV post-administration. Summary/Conclusion: Our results indicated that HLSC-EVs and TKIs have a synergistic anti-tumour effect on renal CSCs inducing an enhancement of apoptosis by a combined effect on intracellular pathways pivotal in the induction of tumour growth and survival. This effect appear to be due to an EV-dependent enhancement of TKI induced mechanisms and not to epigenetic changes induced by EV leading to increased TKI sensitivity. This study provides a rational for a combined use in tumour treatment. Funding: This study was supported by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), project IG2012 and by grant no. 071215 from Unicyte. OT03.02 Exosomal nidogen 1 drives liver cancer metastasis by inducing secretion of tumour necrosis factor receptor 1 from activated lung fibroblasts Xiaowen Mao, Sze Keong Tey and Judy Yam The University of Hong Kong, Hong Kong, Hong Kong Introduction: Hepatocellular carcinoma (HCC) is an aggressive tumour with metastasis as a signature in the advanced stage. Acquisition of migratory and invasive behaviours is fundamental to cancer cells to metastasize. A supportive microenvironment for the colonization of incoming disseminated cancer cells during metastasis is also indispensable. Exosome shedding has emerged as an important channel for intercellular communication in tumour microenvironment during metastasis. Methods: Exosomes derived from HCC cell lines were functionally characterized by in vitro and in vivo assays. Proteomic profiling and expression level of exosomal proteins were analysed by mass spectrometry and enzyme-linked immunosorbent assay (ELISA), respectively. The study of interplay between exosomes, HCC cells and lung fibroblasts were carried out using functional assays, immunofluorescent staining and ELISA. Results: Exosomes derived from metastatic HCC cells augmented cell migration and invasiveness. In animal model, metastatic-exosomes promoted liver tumour formation, increased incidence of distant metastasis to lungs as well as facilitated colonization of hepatoma cells in lungs and enhanced the permeability of pulmonary vasculature. Proteomic profiling of exosomes identified nidogen 1 (NID1) as a functional component responsible for the promoting effect of metastatic-exosomes. Our data showed that suppression of exosomal NID1 (exo-NID1) significantly diminished the biological activities of metastatic-exosomes. Apart from HCC cells, exo-NID1 enhanced the growth and induced activation of lung fibroblasts. Tumour necrosis factor receptor 1 (TNFR1), found to be released by lung fibroblast pretreated with metastatic-exosomes, showed potent effect in promoting HCC cell motility. Notably, the level of exo-NID1 was well correlated with the metastatic potential of parental HCC cells. Encouragingly, the level of NID1 in circulating exosomes of HCC late stage patients was higher than those at early stage. Summary/Conclusion: Our study reveals the novel role of NID1, in the form of exosomes, in HCC metastasis and illuminates the expression profile of exosomal NID1 with clinical significance. Our study implicates that targeting signalling pathway mediated by exosomes of metastatic HCC as a therapeutic strategy for HCC. Funding: RGC GRF (17,113,116) and SKLLR IRF 2017. OT03.03 Cancer extracellular vesicles create functional heterogeneity of cancer-associated fibroblasts in gastric cancer Yutaka Naito ^a, Yusuke Yamamoto^b, Akiko Kogure^c, Iwao Shimomura^d, Minami Kumazaki^c and Takahiro Ochiya^d ^aDivision of Molecular and Cellular Medicine, National Cancer Center Research Institute, London, UK; ^bDivision of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, USA; ^cDivision of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan; ^dDepartment of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Shinjyuku-ku, Japan Introduction: Cancer-associated fibroblasts (CAFs) are the major stromal components in the various types of malignancies. It has been recognized that the functional heterogeneity of CAFs provide an appropriate microenvironment for tumour progression. However, it is still largely unknown how functional heterogeneity of CAF is governed by tumour cells. In this study, we investigated the role of extracellular vesicles (EVs) on the formation of CAF functional heterogeneity. Methods: We treated EVs derived from high-metastatic diffuse-type gastric cancer (DGC) cells or low-metastatic DGC cells to the fibroblasts. By comparing transcriptome profiles of fibroblasts with the EVs, we sought to understand how high-metastatic DGC cells created an appropriate microenvironment for the metastasis. Results: Our whole-transcriptome analysis of fibroblasts revealed that high metastatic DGC cell-derived EVs strongly induced the expression of inflammatory chemokines such as CXCL1 and CXCL8. However, it is not observed in the fibroblast treated with EVs from low-metastatic DGC. Interestingly, both cancer-derived EVs did not affect the expression of alpha-smooth muscle actin (α-SMA), a typical marker of myofibroblast phenotype. When fibroblasts were treated with TGFβ, α-SMA was clearly induced but suppressed inflammatory chemokine expression in the fibroblasts. Immunocytochemical analysis of CXCL8 and α-SMA showed distinct populations of activated fibroblasts in the co-culture with high-metastatic DGC, suggesting that functional heterogeneity was generated by EVs. We also identified that various miRNAs including miR-193b were enriched in high metastatic DGC cell-derived EVs to induce chemokine expression in fibroblasts. Summary/Conclusion: Our findings suggest that the intercellular crosstalk of high-metastatic DGC and fibroblasts via EVs contributes to forming the appropriate tumour microenvironment toward the metastasis. OT03.04 Extracellular vesicles from obese human adipose tissue alter the invasive and proliferative properties of prostate cancer cells Anca D. Dobrian ^a, Ryan Huyck, Bronson Haynes, Vanessa Correll, Avennette Pinto, James Reed, Matthew Freedman, William McPheat, Julius O. Nyalwidhe^b, O. John Semmes^b ^aEastern Virginia Medical School, Norfolk, USA; ^bLeroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, USA Introduction: Obesity increases the risk and aggressiveness of multiple cancers including prostate cancer. Adipose tissue (AT) is a rich source of extracellular vesicles (EVs) that were shown to contribute to vascular and metabolic pathologies. Here we characterized the miRNA and proteome of EV isolated from human visceral (V) and subcutaneous (S) fat of bariatric subjects and explored their mechanistic effects on molecular and functional phenotypes of metastatic prostate cancer cells. Methods: Paired S and V AT collected intraoperatively were used to isolate EVs by ultracentrifugation (n = 27). DIO-labelled EV-S or EV-V was incubated overnight with PC3-ML metastatic prostate cancer cells. EV uptake, proliferation, migration and invasion were quantified by fluorescence microscopy, BrdU incorporation, wound healing and invasion assays, respectively. The miRNA and proteome cargo of EVs were measured using the Nanostring platform and LC/MS/MS. Changes in gene expression in recipient PC3-ML cells were determined using Nanostring. Results: EV-S and EV-V produced similar effects on recipient PC3-ML cells. EVs increased cell proliferation by ~1.8-fold (p < 0.05); had no effect on cell migration but dramatically decreased cell invasion by 2.5-fold (p < 0.01) compared to untreated controls. Gene expression in recipient PC3-ML cells showed significant two to three fold decrease in expression of 8 MMPs without changes in TIMP expression. Mesenchymal markers Snail and Zeb were also significantly decreased and seven glycolytic and PPP enzymes were 1.5- to 2.5-fold increased. Consistent with these changes, the miRNA cargo of EVs was shown to target all the above pathways and the top pathways detected in the EV proteome were metabolism and energy production. Summary/Conclusion: AT EVs appear to induce a mesenchymal to epithelial transition in prostate cancer cells. This study reveals a novel role of EVs from human AT on metastasis and suggests a new mechanistic link between obesity and prostate cancer. Funding: Commonwealth of Virginia Health Research Board. OT03.05 Novel vesicular mediators of peritoneal metastases Shelly Loewenstein ^a, Fabian Gerstenhaber^b, Nir Lubezky^b, Eran Nizri^b, Joseph Klausner^b, Noam Shomron^c, Guy Lahat^b ^aTel Aviv Sourasky Medical Center, Tel Aviv, Israel; ^bSurgery Division, Tel Aviv Sourasky Medical Center, Tel-Aviv, Israel; ^cTel Aviv university, Tel Aviv, Israel Introduction: Malignant progression results from a dynamic crosstalk between stromal and cancer cells. Recent data suggest that this crosstalk is mediated by exosomes, nanovesicles secreted by various cell types which allow the transfer of proteins, and nucleic acids between cells. We investigated the potential role of omental fat exosomes in gastric cancer peritoneal metastasis. Methods: Omental fat exosomes were produced from fresh human omental fat specimens. Proliferation, migration, invasion and chemoresistance were used to evaluate the phenotypic behaviour of omental-exosomes treated gastric cancer cells. Using a comprehensive cytokine array, we identified the proteome of omental-exosomes. Exosomal miRNAs were profiled using NanoString technology. A xenograft model was used to evaluate in vivo effects of omental-exosomes on gastric cancer tumour growth. Results: Initially, we demonstrate a robust uptake of omental fat exosomes by gastric cancer cells. We show that these exosomes enhance gastric cancer cell proliferation, migration and invasion. We also revealed that the number of exosomes is directly related to their effect on gastric cancer cells. We further show that omental fat exosomes induce gastric cancer cellular chemoresistance to platinum-based therapy, and that omental exosomes augment gastric cancer xenograft tumour growth in-vivo. Using a cytokine array, we characterized the proteome of omental fat exosomes compared to SC exosomes. miRNA profiling identified several established oncomiRs. These vesicles carry numerous proteins and miRNAs implicated in cellular adhesion and chemotaxis, tumour growth and motility as well as chemoresistance; some of these molecules have been reported as pro-tumourigenic factors in gastric cancer. Finally, we demonstrate that omental fat-exosomes increase the expression of transcription factors, mRNA of extracellular matrix proteins and adhesion molecules within gastric cancer cells. Summary/Conclusion: These observations demonstrate for the first time the uptake of omental fat exosomes by cancer cells; these vesicles carry different molecules which promote gastric cancer cellular aggressiveness in vitro and in vivo. Taken together, our data imply that omental fat exosomes might play a role in gastric cancer peritoneal spread. OT03.06 Non-SUMOylated Cx43 changes the recruitment of cellular components into exosomes switching the role of these vesicles in metastatic melanoma Adrián Varela-Vázquea^a, María D. Mayán Santos ^b, Amanda Guitián-Caamaño^c, Alejandro Castro-Iglesias^d, Tamara Camino Martínez^e, Susana B. Bravo-López^f, María Pardo^g, Teresa Calleja-Chuclá^h and Eduardo Fonseca^c ^aCellCOM research group. Instituto de Investigación Biomédica de A Coruña (INIBIC). Servizo Galego de Saúde (SERGAS), A Coruña, Spain; ^bTranslational Research in Cell Communication and Signalling (CellCOM). Instituto de Investigación Biomédica de A Coruña (INIBIC). Xubias de Arriba, 84 15006 A Coruña, Spain., A Coruña, Spain; ^cCellCOM research group. Instituto de Investigación Biomédica de A Coruña (INIBIC). Servizo Galego de Saúde (SERGAS), A Coruña, Spain; ^dTranslational Research in Cell Communication and Signalling (CellCOM). Instituto de Investigación Biomédica de A Coruña (INIBIC), A Coruña, Spain; ^eGrupo Obesidómica.Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS). Complejo Hospitalario Universitario de Santiago. Travesía da Choupana s/n. 15706, Santiago de Compostela, Spain, Santiago de Compostela, Spain; ^fProteomics laboratory. Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Complexo Hospitalario Universitario de Santiago de Compostela (CHUS), A Coruña, Spain; ^gGrupo Obesidómica. Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS). Complejo Hospitalario Universitario de Santiago. Travesía da Choupana s/n. 15706, Santiago de Compostela, Spain, Santiago de Compostela, Spain; ^hServicio de Farmacia Hospitalaria. CH-Universitario A Coruña (XXIAC). Servizo Galego de Saúde (SERGAS), Universidade da Coruña. Xubias de Arriba, 84 15006 A Coruña, Spain., A Coruña, Spain Introduction: Connexin43 (Cx43), a transmembrane protein involved in cell communication and signalling, has been described as a tumour suppressor factor in melanoma, however its role in disease progression remains under debate. Extracellular vesicles (EVs) released by melanoma cells provide signals and “educate” distant cells. The presence of Cx43 in EVs provides these particles with an additional capacity to exchange small molecules such as RNAs, metabolites or ions with target cells via gap junction channels (GJs).In this study, we have investigated the role of exosomal Cx43 in metastatic melanoma. Methods: Protein levels and activity were studied by western-blot, immunofluorescence, colony formation and proliferation and migration assays. GJIC by Scrape loading. EVs were isolated by ultracentrifugation and analysed using the NanoSight and electron microscopy. Their content was analysed by mass spectrometry (MS) and by RNA-seq. Results: Low levels and SUMOylated Cx43 in BRAF-mutant human melanoma cells was associated with cytoplasmic distribution and low incidence of dye coupling (GJIC). Ectopic Cx43 gene expression using vectors restored Cx43 membrane localization, raised GJIC and increased Cx43 in the EVs. EVs isolated from BRAF-mutant melanoma cells overexpressing Cx43 only contains the non-SUMOylated Cx43. When different melanoma cell lines were exposed to exosomes containing Cx43, these EVs significantly decreased cell proliferation and blocked colonies growth. The effect of exosomal Cx43 was compared to the overexpression of the protein. The presence of Cx43 in EVs significantly increased the sensitivity of BRAF-mutant metastatic melanoma to drugs such as BRAF/MEK inhibitors. The RNA and proteomic component identified by RNA-Seq and MS revealed that exosomal Cx43 through its scaffolding function could be involved in the recruitment of proteins and small RNAs to the EVs switching the messages and therefore the role of these EVs in melanoma. Summary/Conclusion: Our results indicate that exosomal particles containing Cx43 are potent vehicles to combat metastatic melanoma. Further understanding of the role of Cx43 in EVs will have implications for the development of new therapeutic strategies. For instance, we demonstrated their ability as drug carriers to combat metastasic melanoma when these vesicles contain Cx43. Symposium Session 4: EV Biogenesis I Chairs: Nobuyoshi Kosaka; Clotilde ThéryLocation: Level B1, Hall A 11:00–12:30 OT04.01 Linking the trafficking of CD63 and CD9 to their secretion mechanisms into extracellular vesicles Mathilde Mathieu ^a, José Ignacio Valenzuela^b, Mathieu Maurin^a, Mabel Jouve^a, Nathalie Nevo^a, Gaëlle Boncompain^a, Franck Perez^b and Clotilde Thery^c ^aInstitut Curie, INSERM U932, Paris, France; ^bInstitut Curie, umr144, Paris, France; ^3Institue Curie, Paris, France Introduction: A major challenge in the study of extracellular vesicles is to characterize and separate the different extracellular vesicle (EV) subtypes of a different origin. Indeed, small EVs from the plasma membrane or from endosomes cannot be separated with the classical EV isolation methods. Moreover, even if some of their molecular mechanisms of secretion are known, it is challenging to find specific mechanisms for one particular subtype (see perspective article: Mathieu et al. Nat cell Biol 2019, in press). Understanding how markers of subtypes of EVs are directed to similar or different EVs could help to differentiate them, eventually to describe their specific functions. At least two different populations of small EVs were previously described, one carrying the three tetraspanins CD63, CD9 and CD81, and one with CD9 only (Kowal et al. PNAS 2016). Methods: We chose to study in HeLa cells the trafficking of CD63 and CD9 and its link with their secretion in EVs, using the RUSH system to synchronize and follow their post-Golgi trafficking (Boncompain et al. Nat Methods 2012). We used the RUSH system to perform live-cell imaging, electron microscopy, immunofluorescence and flow cytometry analyses at different steps of trafficking, and to analyse EVs secreted after a specific time of trafficking. Results: Despite their presence in the same EVs, CD63 and CD9 do not traffic to the same final compartments. While CD63 is endosomal, CD9 is located on the plasma membrane. We showed that CD9 could be found transiently with CD63 in intracellular compartments before reaching the plasma membrane (PM), while CD63 goes to the PM before being internalized. By forcing stable expression of CD63 at the PM, or impairing post-Golgi and endosomal trafficking, we observed increased secretion of CD63+ but not CD9+ EVs. Summary/Conclusion: Our results demonstrate that small EVs can form both at the PM and inside multivesicular endosomes. Our tools can be used to determine the respective effects of drugs and gene silencing on secretion of each of these EVs OT04.02 Interdependency of the multiple endosomal sorting mechanisms influencing exosome biogenesis Roberta Palmulli^a, Guillaume van Niel^b, Frederik Verweij^b, Xavier Heilingenstein^a, Eric Rubinstein^c and Graça Raposo^a ^aInstitut Curie, PSL Research University, CNRS UMR144, Paris, France; ^bCPN, Centre for Psychiatry and Neuroscience, Hôpital Saint-Anne, Universite´ Descartes, INSERM U894, Paris, France; ^cInserm U935 (ex. U1004) – Paul Brousse Hospital – André Lwoff Institute, Villejuif, France Introduction: Exosomes are generated as intraluminal vesicles (ILVs) in the multivesicular endosome (MVE). In the endosomal system, protein cargoes either are sequestered to ILVs by inward budding or exit the system by outward budding. Sorting to ILVs is mediated by various machineries, whose interdependency is poorly understood, and is likely counterbalanced by recycling mechanisms that retrieve protein from MVEs. We have taken profit of the particular role of CD63 in the balance between ESCRT-dependent and -independent biogenesis of ILVs and in the sorting of ApoE in melanoma cells to elucidate the interdependency of different sorting mechanisms influencing exosome composition. Methods: After siRNA depletion of reported key actors of exosome production, EVs released by melanoma cells were isolated by differential ultracentrifugation and floatation on density gradient and characterized using biochemistry and electron microscopy. ILV biogenesis and sorting of specific cargoes throughout the endosomal system was assessed by immunofluorescence or electron microscopy after high-pressure freezing. Results: Our data show that melanoma cells secrete subpopulations of exosomes with different density and composition. Investigation of known key regulators of in- or outward budding in MVEs differently affected exosome subpopulations. In particular, CD63 modulates ApoE secretion on exosomes and its cellular localization, suggesting that CD63 is a master regulator of cargo trafficking in the endosomal system. Summary/Conclusion: Our data highlight that exosomes biogenesis is not only dependent on ILV budding but also on a global regulation of endosomal homeostasis. Our study provides a better perception of the interconnections existing between sorting of cargoes to ILVs and their retrieval from the endosomal system. This broader view is crucial to understand the precise roles of reported regulators of exosomes biogenesis that are broadly used by the community. OT04.03 A bright, versatile live cell reporter of exosome secretion and uptake Bong Hwan Sung ^a and Alissa Weaver^b ^aVanderbilt University, Nashville, USA; ^bDepartment of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, USA Introduction: Small extracellular vesicles (EVs) called exosomes affect a variety of autocrine and paracrine cellular phenotypes. Understanding the function of exosomes in these processes requires a variety of tools. We previously constructed a live-cell reporter, pHLuorin-CD63 that allowed dynamic monitoring of exosome secretion in migrating and spreading cells. However, there were some caveats to its use, including relatively low fluorescent expression in cells and the inability to make cell lines that stably express the protein. Methods: By incorporating a stabilizing mutation in the pHLuorin moiety, M153R, pHLuorin-CD63 now exhibits higher and stable expression in cells and superior monitoring of exosome secretion. Cancer cells stably expressing pHLuorin_M153R-CD63 were imaged using a variety of microscopy techniques including a confocal and wide-field microscopy and a correlative light-electron microscopy. Results: pHLuorin_M153R-CD63 was exclusively detected in exosome-enriched small EV preparations. Live-cell imaging revealed pHLuorin_M153R-CD63-positive puncta left behind migrating cells suggesting the deposition consists of exosomes. Those puncta and trails were not only positive for other exosome markers such as Alix and TSG101 but also correspond to small EVs observed by a scanning electron microscope. In addition, follower cells exhibited pathfinding behaviour over pHLuorin_M153R-CD63 deposits. Incorporation of mScarlet, a non-pH-sensitive red fluorescent tag, to pHLuorin_M153R-CD63 further improves the ability to track trafficking and secretion of multivesicular bodies (MVBs) in cells allowing visualization of trafficking to the leading edge of migrating cells and uptake of external exosome deposits. Summary/Conclusion: Using pHLuorin_M153R-CD63 construct, we demonstrate superior visualization of exosome secretion in multiple contexts and identify a role for exosomes in promoting leader-follower behaviour in collective migration. By incorporating a further non-pH-sensitive red fluorescent tag, this reporter allows visualization of the entire exosome lifecycle, including MVB trafficking, exosome secretion, exosome uptake and endosome acidification. This new reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes. OT04.04 An explanation for “PS-negative” extracellular vesicles: endogenous annexin-a5 from the cytosol cover externalized phosphatidylserines on plasma membranes Anis Khiat, Dominique Charue, Sihem Sadoudi, Sylvain Le Jeune, Marie Lê-Hoang, Chantal Boulanger, Olivier P. Blanc-brude INSERM ‘ParCC’ Paris-Cariovascular Research Center, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, and Université Sorbonne, Paris, France Introduction: Stressed cells shed extracellular vesicles (EVs) thought to bear externalized phosphatidylserine (PS) at their surface and promote inflammation, coagulation and tissue injury. Conversely, endogenous cytosolic annexins, such as annexin-A5, orchestrate vesicle trafficking and membrane repair within multiple cell types, via Ca2+-dependent binding to intracellular PS. We hypothesized that endogenous annexin-A5 binds to PS during vesiculation and gets externalized with PS at the surface of EVs. Methods: We purified healthy plasma and red blood cells and induced Ca2+-mediated vesiculation. We assessed annexin-A5 and EV distribution in supernatants by Western blots, FACS, ELISA, cryo-TEM. Results: (1) About 20% cytosolic annexin-A5 leaked out during vesiculation, but cytoskeletal proteins were not released. (2) We separated supernatant EVs from “free” proteins by size-exclusion chromatography and quantified EV-bound vs. “free” annexin-A5. All annexin-A5 remained bound to EVs. Other cytosolic proteins (haemoglobin) bound to EVs only partly. FACS with anti-annexin-A5 antibodies revealed the presence of annexin-A5 at the EV surface. (3) We measured EV-bound and “free” annexin-A5 in plasma, vs PS-, PS+, CD235a+ and annexin-A5+ EVs, and made similar observations. Our study suggests that endogenous annexin-A5 can cover externalized PSs on EVs in the presence of Ca2+. Summary/Conclusion: This new mechanism of PS-neutralization may explain previous reports of apparently “PS-negative” EVs. Conventional detection of EVs with EXOgenous fluorescent annexin-A5 (FACS) may thus depend on PS not being engaged by endogenous annexin-A5 prior to detection. The physiopathological relevance of endogenous PS neutralization may complement enzyme- and ATP-mediated internalization of PS in healthy cells. PS neutralization may become critical when internalization mechanisms are overwhelmed, and serve to restrain PS-mediated reactions and enforce anti-inflammatory and anti-thrombotic control when the integrity of a few cells only is compromised. On the other hand, dysfunctional annexin-A5 or calcium metabolism may contribute to the release of pro-inflammatory and pro-thrombotic PS+ EVs. Funding: Funded by Fondation pour la Recherche Médicale and Sorbonne University. OT04.05 Identification of EV secretion-associated gene involved in melanoma progression by microRNA-based screening Nobuyoshi Kosaka ^a, Fumihiko Urabe^b, Tomofumi Yamamoto^c, Yurika Sawa^d and Takahiro Ochiya^a ^aDepartment of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Shinjyuku-ku, Japan; ^bDivision of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan; ^cDepartment of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan; ^dDepartment of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan Introduction:It has been shown that extracellular vesicles (EVs) derived from cancer cells dictate their surrounding microenvironmental cells or distant cells in the future metastatic organs for the benefit of cancer cells. Thus, revealing the molecular mechanisms underlying the production of EVs would prove to be a valuable contribution for establishing EV-targeted therapy against cancer. However, the precise mechanism of EV production, especially in cancer cells, remains unclear. Here, we established a microRNA-based screening system to identify the molecules involved in EV production from melanoma cells. Methods: Melanoma cell lines, A375 cells, were used in this study. Combined with the ultra-sensitive EV detection method (Yoshioka), ExoScreen, we have screened nearly 2000 miRNAs in melanoma cells. To confirm the results of ExoScreen, we employed the nanoparticle tracking analysis. Target genes of miRNAs were identified by the combination of gene expression analysis and target prediction bioinformatics. Results: miRNAs which suppressed the secretion of EVs from melanoma cells were identified after the screening of nearly 2000 miRNAs. To understand the molecular mechanisms mediated by these miRNAs, the target genes of these miRNAs were identified and evaluated for their contribution to EV production in cancer cells. Indeed, attenuation of these target genes declined the secretion of EVs from melanoma cells, suggesting the contribution of these genes in EV production/secretion. Furthermore, the expression of these genes was higher in melanoma tumour tissues compared with that in normal tissues. Summary/Conclusion: These findings suggest that the miRNAs and their target genes were involved in EV production/secretion, resulting in the promotion of cancer progression. OT04.06 Distinct mechanisms of microRNA sorting into cancer cell-derived extracellular vesicle subtypes Morayma Temoche-Diaz^a, Matthew Shurtleff^a, Ryan Nottingham^b, Jun Yao^b, Alan Lambowitz^b, Randy Schekman^a ^aUniversity of California, Berkeley, Berkeley, USA; ^bUniversity of Texas, Austin, Austin, USA Introduction: Extracellular vesicles (EVs) encompass a variety of vesicles secreted to the extracellular space. EVs have been implicated in promoting tumour metastasis but the molecular compositions of tumour-derived EV sub-types and the mechanisms by which molecules are sorted into EVs remain mostly unknown. As such dissecting different EV sub-populations and analysing the molecular mechanisms behind active cargo sorting is needed. Methods: The highly metastatic breast cancer cell line, MDA-MB-231, was used as the model cell line for this study. Iodixanol linear gradient allowed for the separation of EV sub-populations. miRNA profiling and TGIRT-sequencing was used to study the miRNA content of the distinct EV sub-populations. Cell fractionation and cell-free miRNA packaging reconstitutions, coupled with in vivo confirmation, in cultured cells, were used to study the molecular mechanisms of miRNA sorting. Results: We found that at least two distinct EV sub-populations are released by MDA-MB-231 cells. Their differential biochemical properties suggest different sub-cellular origins (endosomes vs. direct budding from the plasma membrane). Moreover, they are governed by distinct mechanisms of miRNA sorting (active vs. passive). By using biochemical and genetic tools, we found that the Lupus La protein is responsible for mir122 sorting into EVs in vitro and in vivo. Moreover, in vitro studies showed that the Lupus La protein interacts with mir122 with very high affinity. Finally, we uncovered the mir122 motifs required for mir122-La high affinity interaction, and therefore mir122 sorting into EVs. Summary/Conclusion: Two EV sub-populations with distinct sub-cellular origins, are released by MDA-MB-231 cells. Their differential sub-cellular origin is coupled with two distinct mechanisms of miRNA sorting. The Lupus La protein is responsible for the active sorting of mir122 into EVs in vitro and in vivo. Funding: Howard Hughes Medical Institute (HHMI). Oral with Poster Session 1 Chairs: Uta Erdbrügger; Kenneth WitwerLocation: Level B1, Hall B 13:30–15:00 OWP1.01=PS10.10 miR-1227 alters extracellular vesicle shedding Andrew R. Chin ^a, Minyung Kim^a, Valentina R. Minciacchi^b, Tatyana Vagner^a, Javier Mariscal^a, Cristiana Spinelli^a, Mandana Zandian^a, Paolo Gandellini^c, Nadia Zaffaroni^c, Shivani Sharma^d, Sungyong You^a and Dolores Di Vizio^a ^aCedars Sinai Medical Center, West Hollywood, USA; ^bCedars Sinai Medical Center, Frankfurt, Germany; ^cFondazione IRCCS Istituto Nazionale Tumori, Milan, USA; ^dUniversity of California, Los Angeles, Los Angeles, USA Introduction: Extracellular vesicles (EVs) play a key role in cancer development and metastasis by influencing the behaviour of the primary tumour and by aiding the establishment of a pre-metastatic niche in distant organs. This process is due to the EV-mediated functional transfer of biologically active molecules including microRNA (miRNA). miR-1227 is a poorly characterized miRNA that is enriched in EV secreted by prostate cancer (PC) cells in comparison to non-tumorigenic prostate epithelial cells. However, the role of miR-1227 in cancer is poorly understood. Our objective is to determine the role of miR-1227 in PC. Methods: RNA sequencing from miR-1227 stably expressing PC cells, RISCTRAP Immunoprecipitation of miR-1227 bound mRNA and five different in silico miRNA target prediction methods were used to identify putative miR-1227 targets. Exosomes and large oncosomes (LO) were isolated by differential ultracentrifugation followed by density gradient purification. Atomic force microscopy and TRPS were used to quantify exosomes and LO secreted by PC cells stably expressing miR-1227 or vector control. Results: A comparative analysis between different EV subtypes indicates that miR-1227 is enriched in LO, a class of EV that are secreted by highly invasive and metastatic amoeboid-migrating cells. LO carry more RNA than the more widely studied exosomes indicating that LO may be a more robust source of EV-encapsulated miRNA. Gene ontology analysis from miR-1227 targets identified by RNA sequencing from miR-1227 stably expressing PC cells, RISCTRAP Immunoprecipitation of miR-1227 bound mRNA, and in silico miRNA target prediction highlighted several genes related to EV secretion. miR-1227 alters the localization of exosome and LO markers in multiple cancer cell lines, and induces the shedding of LO while inhibiting the shedding of exosomes. Furthermore, miR-1227 induces the migration of poorly migratory cancer cells and increases the expression of tumour supportive cytokines. Summary/Conclusion: Together these data hint that miR-1227 may promote prostate cancer progression through several mechanisms including alteration of EV shedding. Funding: 2017–2022 R01CA218526. 2018–2020 Chesapeake Urology Associates Sanford J. Siegel, MD Prostate Cancer Research Scholarship 2018–2020 Luke Wu-Jei Chang Discovery Fund 2016–2019 PI DoD PCRP Award [22]PC150836 OWP1.02=PF11.14 MSC exosome works through a multi-faceted mechanism of action in joint repair Shipin Zhang ^a, Yedan Wang^b, Francis Keng Lin Wong^c, Ming Wang^b, Ruenn Chai Lai^d, James Hoi Po Hui^b, Sai Kiang Lim^d and Wei Seong Toh^a ^aFaculty of Dentistry, National University of Singapore, Singapore, Singapore; ^bDepartment of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; ^cDepartment of Orthopaedic Surgery, Sengkang General Hospital, Singhealth, Singapore,Singapore; ^dInstitute of Medical Biology, Agency for Science, Technology and Research, Singapore, Singapore Introduction: MSC exosome is increasingly accepted as the principal agent that underpins the therapeutic efficacy of mesenchymal stem cell (MSC) in tissue repair. Here, we aim to elucidate the mechanism of action (MoA) of MSC exosome in immunocompetent rat models of osteochondral defect and osteoarthritis (OA). Methods: Exosomes were purified from conditioned medium of human MSCs by size fractionation. Osteochondral defect creation or anterior cruciate ligament transection to induce OA were performed in 72 adult rats. Thereafter, weekly 100-µl intra-articular injections of 100-µg exosome or PBS vehicle were given. Analysis included weight distribution, histology, immunohistochemistry and cytokine assay. Cellular assays using chondrocytes were performed to determine the exosome-activated cellular processes and signalling pathways. Results: We observed that exosome-mediated repair of osteochondral defects was characterized by increased cellular infiltration and proliferation, enhanced matrix synthesis, together with a regenerative M2 macrophage phenotype and a reduction in pro-inflammatory cytokines IL-1β and TNF-α. In OA joints, MSC exosome mediated an early suppression of pain and degeneration with reduced inflammation, followed by sustained proliferation and matrix restoration that led to cartilage and subchondral bone regeneration. Using chondrocyte cultures, we could attribute some of these cellular activities during exosome-mediated joint repair to exosomal CD73-mediated adenosine activation of AKT and ERK signalling. These effects were partially abrogated by wortmannin or U0126, which inhibited AKT and ERK phosphorylation, respectively. The role of exosomal CD73 was confirmed using CD73 inhibitor and theophylline that showed inhibition of exosome-induced AKT and ERK phosphorylation. Summary/Conclusion: Our observations suggest that MSC exosome works through a multi-faceted MoA that involved multiple cellular processes to restore joint homeostasis and promote regeneration. Funding: National Medical Research Council Singapore (NMRC/CNIG/1168/2017 and NMRC/CIRG/1480/2017). OWP1.03=PS03.11 Identification of extracellular vesicles as biomarkers for myocardial infraction by flow cytometry and automated data processing Aleksandra Gasecka ^a, Edwin van der Pol^b, Frank Coumans^c, Kinga Pluta^d, Grzegorz Opolski^d, Krzysztof J. Filipiak^e, Rienk Nieuwland^c ^a1st Chair and Department of Cardiology, Medical University of Warsaw, Warsaw, Poland; ^bAmsterdam UMC, University of Amsterdam, Department of Biomedical Engineering and Physics, Amsterdam, Netherlands; ^cAmsterdam UMC, University of Amsterdam, Laboratory of Experimental Clinical Chemistry, Amsterdam, Netherlands; ^d1st Chair and Department of Cardiology, Medical University of Warsaw, Warsaw, Poland; ^e1st Chair and Department of Cardiology, Medical University of Warsaw, Warsaw, Poland Introduction: Acute myocardial infarction (AMI) is a major cause of death. To diagnose AMI, measuring troponin concentration is the gold standard. Since troponin is unspecific for AMI, novel biomarkers for AMI are urgently needed. After the onset of AMI, platelets, endothelial cells and blood cells release specific extracellular vesicles (EVs). Our aim is to identify these EVs as biomarkers for AMI diagnosis and treatment monitoring. Methods: The study was approved by the medical ethics committee. Venous blood was collected 24 h, 72 h and 6 months after AMI from fasting patients (n = 60, 64.5 ± 10.8 years, 68% male) and healthy controls (n = 30, 57.7 ± 6.6 years, 62% male). Flow cytometry (Apogee A60 Micro) was used to determine plasma concentrations of EVs labelled with antibodies for activated platelets (CD61, CD62p; PEVs), endothelial cells (CD146; EEVs) and red blood cells (CD235a; RBC-EVs). Processing of 1,224 flow cytometry data files was performed using in-house developed, automated software (MATLAB R2018a), enabling flow rate stabilization, diameter and refractive index determination, MESF calibration, fluorescent gate determination and statistics reporting. Results: Between AMI patients and controls, PEV concentrations in plasma were comparable (p = ns), EEV concentrations increased (p < 0.0001) and RBC-EV concentrations decreased (p < 0.0001). Antiplatelet drug ticagrelor decreased concentrations of PEVs (p = 0.03), compared to less potent clopidogrel but did not affect EEVs and RBC-EVs. In turn, concentrations of EEVs, but not PEVs and RBC-EVs, positively correlated with the dose of atorvastatin (p < 0.001). The antioxidative β-blocker carvedilol increased concentrations of RBC-EVs, compared to nebivolol (p = 0.05) but did not affect PEVs and EEVs. Summary/Conclusion: Flow cytometry and automated data processing were used to find biomarkers for AMI based on EVs in plasma. During treatment, ticagrelor decreased PEV concentrations, atorvastatin increased EEV concentrations and carvedilol increased RBC-EV concentrations, suggesting that EVs might be used to monitor AMI treatment. AMI patients differed from controls regarding EEV and RBC-EV concentrations, but not PEVs, likely because blood was collected 24 h after the start of antiplatelet therapy. In follow-up studies, it is crucial to collect blood prior to treatment. OWP1.04=PF11.15 Exosome mediated enhancement of cellular therapy in acute myelogenous leukemia (AML) Theo Borgovan ^a, Peter Quesenberry^b, Mike Deltatto; Sicheng Wen^c, Mark Dooner^b ^aBrown University Department of Hematology Oncology; Rhode Island Hospital, Pawtucket, USA; ^bBrown University Department of Hematology Oncology; Rhode Island Hospital, providence, USA; ^cBrown University/ Rhode Island Hospital, Providence, USA Introduction: Of the AML patients able to tolerate curative therapy with chemotherapy and stem cell transplant many are challenged by treatment related toxicities as well as graft vs. host disease. There is novel work exploring the utility of haploidentical cellular therapy infusion in order to incite purposeful recipient immune response and subsequent cytokine storm to treat refractory AML. Our group has demonstrated the healing potential of bone marrow derived mesenchymal stem cell extracellular vesicles (MSC-EVs) across multiple disease states, most recently demonstrating the pro-apoptoic signalling imparted by these nanoparticles on nascent leukemic cells in vivo; as well as the potentiating effects of MSC-EVs when used as an adjunct to standard cytarabine chemotherapy. We have also shown the protective role of hMSC EV on radiated BM and stem cell recovery. Methods: Kasumi AML cells lines were seeded with MSC-derived EVs. Vesicles were isolated using an established differential centrifugation technique, and were co-cultured with Kasumi cells for various time points. To study cellular viability, we used a fluorescence-based method for quantifying viable cells. We also explored various modes of death EVs may illicit via a tri-dye Abcam assay designed to simultaneously monitor apoptotic, necrotic and healthy cells. Both assays were used to measure viability and apoptosis in similar experiments employing cytarabine Results: AML cell Proliferation Decreased after 1–6 days of co-culture with hMSC-derived EVs. Apoptosis is the primary mode of death induced. AML cell proliferation decreased synergistic after 1–6 days of co-culture with hMSC-derived EVs ± Cytarabine. Summary/Conclusion: MSCs inhibits the proliferation of the AML cell line in vitro and work synergistically with cytarabine chemotherapy to promote apoptotic death in AML cell lines. Our prior work has shown that MSC-EVs can abate the effects of toxic chemo/radiation and serve to protect stem cell allowing for quicker recover in cell blood counts. Based on the innate ability of MSC-EV to directly alter the cellular machinery of abnormal leukemic cell and of nascent immune cells our corollary hypothesis is that BM-derived MSC-EVs may serve as suitable alternative to conditioning chemo/radiation in the AML setting and will enhance the effects seen by cellular therapy infusion. Funding: t32. OWP1.05=PF12.09 Extracellular vesicles derived from amniotic fluid stem cells rescue impaired foetal lung development via the release of microRNAs Lina Antounians, Vincenzo Catania, Benjamin Liu, Areti Tzanetakis, Louise Montalva and Augusto Zani The Hospital for Sick Children, Toronto, Canada Introduction: Incomplete lung development, also known as pulmonary hypoplasia (PH), is a recognized cause of neonatal death. To date, there is no effective treatment that promotes foetal lung growth and maturation. Herein, we describe a stem cell-based approach that enhances foetal lung development via the administration of extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) in rat models of PH. Moreover, we report the microRNAs present in AFSC-EVs that are responsible for these beneficial effects. Methods: AFSC-EVs were isolated by ultracentrifugation from conditioned medium (CM) of c-Kit+ rat AFSC that were grown in exosome-depleted FBS for 18h. AFSC-EVs were assessed for size (nanoparticle tracking analysis), morphology (TEM), and expression of CD63, Hsp70, Flo-1 and TSG101 (Western). Ex vivo: Pregnant dams were gavaged nitrofen at E9.5 to induce foetal PH. At E14.5, foetal lungs were harvested, and incubated with culture medium alone, AFSC-CM, or AFSC-EVs. Foetal lungs from untreated dams served as control. Lungs were compared for terminal bud density and surface area at 72 h, by two independent investigators. In vitro: Foetal rat lung organoids were generated with epithelial cells from normal and hypoplastic lungs. Organoids were cultured for 10 days in either medium alone or medium supplemented with AFSC-EVs. Lung organoids from untreated normal pups served as control. Organoids were assessed for proliferation (Ki67) and markers of epithelial cell differentiation via immunofluorescence. RNA-sequencing: RNA was isolated using SeraMir, constructed into libraries (CleanTag Small RNA) and sequenced on NextSeq High Output single-end sequencing run. Results: Administration of AFSC-EVs increased terminal bud density and surface area of lung explants back to control levels and promoted lung epithelial cell differentiation in lung organoids (increased SPC and CC10 expression). AFSC-EVs contain 901 microRNAs, some of which are crucial for foetal lung development, such as miR17 ~ 92 cluster. Summary/Conclusion: Administration of AFSC-EVs rescues impaired foetal lung development in experimental models of PH. AFSC-EV regenerative ability is exerted via the release of miRNAs some of which regulate genes involved in foetal lung development. AFSC-EVs represent a promising therapeutic strategy for PH in foetuses. Funding: CIHR-SickKids Foundation. OWP1.06=PS01.11 Extracellular vesicles from Fat-laden hypoxic hepatocytes activates pro-fibrogenic signals in Hepatic Stellate Cells Alejandra Hernandez ^a, Yana Geng^b, Daniel Cabrera^c, Nancy Solis^d, Han Moshage^e and Marco Arrese^d ^aPontificia Universidad Católica de Chile; University Medical Center of Groningen, Groningen, Netherlands; ^bUMCG, Groningen, Netherlands; ^cPontificia Universidad Católica de Chile/Universidad Bernardo O´Higgins, SANTIAGO, Chile; ^dPontificia Universidad Católica de Chile, Santiago, Chile; ^eUniversity Medical Center Groningen, Groningen, Netherlands Introduction / Background: Transition from isolated steatosis to non-alcoholic steatohepatitis is a key issue in non-alcoholic fatty liver disease (NAFLD). Recent observations in patients with obstructive sleep apnoea syndrome (OSAS), suggest that hypoxia may contribute to disease progression mainly through activation of hypoxia inducible factor 1α (HIF-1α)-related pathways. Release of extracellular vesicles (EV) by injured hepatocytes may be involved in NAFLD progression. Aim: to explore whether hypoxia modulates the release of EV from free fatty acid (FFA)-exposed hepatocytes and assess cellular crosstalk between hepatocytes and LX-2 cells (human hepatic stellate cell line). Methods: HepG2 cells were treated with FFAs (250 μM palmitic acid + 500 μM oleic acid) and chemical hypoxia (CH) was induced with Cobalt (II) Chloride, which is an inducer of HIF-1α. Induction of CH was confirmed by Western blot (WB) of HIF-1α. EV isolation and quantification was performed by ultracentrifugation and nanoparticle tracking analysis respectively. EV characterization was performed by electron microscopy and WB of CD-81 marker. LX-2 cells were treated with 15 μg/ml of EV from hepatocytes obtained from different groups and markers of pro-fibrogenic signalling were determined by quantitative PCR (qPCR), WB and immunofluorescence (IF). Results: FFA and CH-treatment of HepG2 cells increased gene expression of IL-1β and TGF-β1 in HepG2 cells and increased the release of EV compared to non-treated HepG2 cells. Treatment of LX-2 cells with EV from FFA-treated hypoxic HepG2 cells increased gene expression of TGF-β1, CTGF, α-SMA and Collagen1A1 compared to LX-2 cells treated with EV from non-treated hepatocytes or LX-2 cells exposed to EV-free supernatant from FFA-treated hypoxic HepG2 cells. Moreover, EV from FFA-treated hypoxic HepG2 cells increased Collagen1A1 and α-SMA protein levels. Summary/Conclusion: CH promotes EV release from HepG2 cells. EV from hypoxic FFA-treated HepG2 cells evoke pro-fibrotic responses in LX-2 cells. Further genomic and proteomic characterization of EV released by steatotic cells under hypoxia are necessary to further delineate their role in the crosstalk between hepatocytes and stellate cells in the setting of NAFLD and OSAS. Funding: FONDECYT 1150327–1150311. OWP1.07=PS08.07 Exploration of the surface modification of outer membrane vesicles Maximilian Richter^a, Eleonora Diamanti^b, Anna Hirsch^b, Gregor Fuhrmann^c ^aHelmholtz-Institute for Pharmaceutical Research Saarland, Biogenic Nanotherapeutics, Saarbruecken, Germany; ^bHelmholtz-Institute for Pharmaceutical Research Saarland, Drug Design and Optimization, Saarbruecken, Germany; ^3Helmholtz-Institute for Pharmaceutical Research Saarland, BION, Saarbruecken, Germany Introduction: Introducing bacteria-binding small molecules to the surface of outer membrane vesicles (OMVs) could greatly improve their potential for antimicrobial drug delivery too difficult to treat bacteria. Among the small number of studies on surface modification of OMVs, very few deal with small molecules. The aim of the present study is to evaluate different methods of introducing bacteria specific targeting moieties to OMVs. We assessed the modification of surface proteins using N-hydroxysuccinimide (NHS) esters, well established for mammalian extracellular vesicles (EVs), cholesterol insertion, mainly applied for liposomes, and the novel application of diazo-transfer followed by click-chemistry. Methods: OMVs were obtained from model myxobacteria by differential ultracentrifugation (UC) followed by size-exclusion chromatography (SEC). For cholesterol insertion and NHS ester-modification, purified OMVs were incubated with either cholesteryl PEG 2,000 FITC or sulfo cyanine7 NHS ester. For diazo transfer the pellet after UC was incubated with a diazo transfer agent and the OMVs subsequently conjugated with DBCO-AF594. Unincorporated dye was removed by SEC. Liposomes were composed of DMPC and DPPC in 2:3 molar ratio. Results represent correlated fluorescence intensity and particle number. Results: Treatment with sulfo cyanine7 NHS ester led to the modification with 547 ± 163 molecules per OMVs, compared to 18 ± 1 for the control using sulfo cyanine7 acid. Cholesterol insertion introduced 4 ± 1 molecules per OMV, compared to 101 ± 23 for liposomes. First results for the diazo-transfer showed 71 dye-molecules per OMV, with 32 for the control. Summary/Conclusion: Of the three methods, NHS ester-modification displayed the highest efficiency, similar to published results for mammalian EVs. In comparison, diazo transfer only yielded ~13% of the dye-molecules per particle. However, there are still many parameters to be optimized for this method, including OMV concentration and incubation period. Cholesterol insertion was unsuccessful for OMVs, probably owing to their membrane structure. In this study, we aim to get important insights into the modification of OMVs for bacterial targeting and EV-surface engineering in general. Funding: This project was funded by Studienstiftung des Deutschen Volkes and Bundesministerium fuer Bildung und Forschung. OWP1.08=LBT02.03 Isolation of neuron-specific extracellular vesicles Dmitry Ter-Ovanesyan ^a, Maia Kipman^b, Emma Kowal^c, Ju Hyun Lee^b, Wendy Trieu^b, Aviv Regev^d, David Walt^b and George Church^b ^aHarvard, Cambridge, USA; ^bWyss Institute, Boston, USA; ^cMIT, Cambridge, USA; ^dBroad Institute, Cambridge, USA Introduction: Human biological fluids contain extracellular vesicles (EVs) from different cell types. It would be incredibly useful to be able to isolate EVs that originated from specific cell types for diagnostic purposes as a way to gain molecular information (RNA, protein) from inaccessible cell types non-invasively. Methods: We have developed a general framework for identifying EV surface markers that can be used for immuno-isolation of cell type specific EVs. As a proof of principle, we have applied this framework to the isolation of neuron-derived EVs from human cerebrospinal fluid or plasma. In addition to the computational analysis, we have developed an in-vitro system of human neurons differentiated from human induced pluripotent (iPS) cells. We performed mass spectrometry on EVs isolated from these neurons to identify neuron-specific proteins. We also used this system to develop a robust immune-isolation method for neuron EV markers. Results: We have characterized the proteins present in neuron exosomes by mass spectrometry and then used computational analysis of published gene expression and proteomics data to come up with a list of candidate neuron-specific EV markers. After developing methods for immuno-isolation of neuron EVs with these markers, we applied our methods to human cerebrospinal fluid and plasma. Summary/conclusion: We have developed a framework for the isolation of cell type specific EVs through the combination of an experimental in vitro system and computational analysis of gene expression and proteomics data. We have applied this framework to the isolation of neuron-specific EVs in human biological fluids. We envision these methods being broadly applicable to the development of novel diagnostic biomarkers for a variety of diseases. OWP1.09=LBT01.01 Coagulation influences properties of extracellular vesicles isolated from autologous blood derived products Andrea De Luna ^a, Alexander Otahal^a, Olga Kuten^b, Zsombor Lacza^c and Stefan Nehrer^a ^aDanube University Krems, Krems, Austria; ^bOrthoSera GmbH, Krems, Austria; ^cOrthosera GmbH, Krems, Austria Introduction: Platelet rich plasma (PRP) is the most commonly used blood derivative in clinics due to its high concentration of platelets and perceived high growth factor levels. Drawbacks of using PRP are discrepancies among preparation protocols and the presence of cells (platelets, leucocytes) which can evoke cellular processes (e.g. inflammation) when injected into the host. One possibility is to isolate only the active components of blood derivatives which may overcome this problem. In the current study, we focused on extracellular vesicles (EVs) isolated from two autologous blood derivatives, PRP and hyperacute serum and investigated whether the clotting cascade influences EV properties. Methods: EVs were isolated from citrate-anticoagulated PRP (CPRP) and hyperacute serum using differential ultracentrifugation followed by a size exclusion chromatography. Particle concentration and size were determined by nanoparticle tracking analysis (NTA). Cryo-electronmicrosopy was performed to visualize isolated EVs. Expression of miRNAs transported within EVs as well as in their respective input material was analysed by qPCR. Results: NTA revealed higher particle concentrations and bigger sized EVs within CPRP compared to hyperacute serum. These findings were confirmed by cryo-electronmicroscopy. Profound differences were detected regarding miRNA expression between the two blood derivatives. In total, 126 miRNAs were identified which were expressed both in input material as well as in the corresponding EVs. The correlation between miRNAs in EVs and input material was higher in CPRP compared to hyperacute serum meaning that in hyperacute serum miRNAs were identified which were higher expressed in EVs than in the corresponding input material. Summary/conclusion: EVs from autologous blood products represent a novel and cell-free regeneration approach. We observed that the clotting cascade (plasma versus serum) has an influence on concentration, size and miRNA expression patterns of EVs. These differences might have an impact on the biological mode of action of blood-derived products used in clinics. Funding: Financial support was received from the European Fund for Regional Development (EFRE) and the Science Fund of Lower Austria. miRNA expression analysis was performed by TAmiRNA GmbH. Cryo-electronmicroscopy was conducted at the Core Facility of the Vienna Bio-Center. OWP1.10=LBF02.01 Type-2 transglutaminase affects calcium homeostasis in neurons and is released in association with astrocytes-derived exosomes Elisa Tonoli ^a, Ilaria Prada^b, Claudia Verderio^c and Elisabetta Verderio^a ^aNottingham Trent University, Nottingham, United Kingdom; ^bCNR Institute of Neuroscience, Milan, Italy., Milan, Italy; ^cCNR Institute of Neuroscience, Milan, Italy, Milan, Italy Introduction: Type-2 transglutaminase (TG2) has been linked to calcium (Ca2+) dysregulation in conditions such as neurodegeneration. Recent evidences suggest that extracellular vesicles (EVs) contribute to the onset and progression of neurological diseases, and we have recently shown that TG2 is a cargo of EVs in biological fluids (Furini et al., 2018). Here, we hypothesise that TG2 could be released by EVs, interact with neurons and affect neuronal Ca2+ homeostasis. Methods: Primary hippocampal neurons were established from E18 rat embryos. Extracellular TG2 was modulated in neurons either by lipofectamine transfection of a TG2-EGFP construct or by addition of purified TG2. Intracellular Ca2+ concentration ([Ca2+]i) was assessed by live imaging in fura-2/AM-loaded neurons. EVs were isolated from primary astrocytes (60 DIV) by serial centrifugation, characterised by western blotting (flotillin-2 and alix) and nanoparticle tracking analysis (ZetaView). Experiments to assess TG2 influence on exosomes-to-neural cells interactions, using a Renilla sensor based on miR-146a-5p-transfer, are still ongoing. Results: Increase of extracellular TG2 levels in neurons induced an influx of extracellular Ca2+ ions, leading to a significant raise in basal [Ca2+]i both in normal conditions (ΔF340/380 = 0.126 ± 0.014; N = 23; p < 10–5) and with inhibited synaptic transmission (tetrodotoxin) (ΔF340/380 = 0.058 ± 0.005; N = 33; p < 10–5). Nifedipine, a blocker of L-type voltage-operated Ca2+ channels (VOCCs), partially prevented TG2-dependent Ca2+ response (average inhibition 36%; N = 21; p < 10–5), suggesting that Ca2+ influx may occur through L-type VOCCs. To identify the source of extracellular TG2, we analysed EVs isolated from rat primary astrocytes, previously reported to release TG2 into the matrix especially in inflammatory conditions. TG2 was detected in astrocytic exosomes (but not in ectosomes) only upon LPS stimulus, and not in the EVs-free medium, suggesting that TG2 is a cargo of exosomes during neuroinflammation. Summary/conclusion: TG2 is externalised through astrocyte-derived exosomes upon neuroinflammatory stimuli. Extracellular TG2 mediates the opening of L-type VOCCs in neurons and sets basal [Ca2+]i at higher levels, which could have a significant impact on neuronal activity in neuroinflammation. Funding: John Turland PhD bursary (NTU) and IBRO travel fund. OWP1.11=LBT01.02 Ev-avogadro project: towards a liposomal concentration standard for extracellular vesicle research Gergo Barta^a, Diana Kitka^a, Andras Wacha^a, Judith Mihaly^a, Attila Bota^a, Krisztina Nemeth^a, Pal Szabo^a, Jean-Luc Fraikin^b and Zoltan Varga ^c ^aResearch Centre for Natural Sciences HAS, Budapest, Hungary; ^bSpectradyne LLC, Torrance, USA; ^cResearch Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary Introduction: There is an unmet need for standardization of concentration measurements in the field of extracellular vesicles (EVs). Liposomes may serve an ideal reference system for EVs, but the determination of the number concentration of liposomes from first principles was not attempted so far. Inspired by the International Avogadro project, we aimed to determine the concentration of liposomes with well-defined size and composition via counting the number of phospholipid molecules in these “nanospheres”. Methods: Liposomes composed of phosphocholine and phosphoglycerol were prepared by the extrusion method. Wide-angle X-ray scattering (WAXS) was used to determine the area-per-lipid value. The size distribution of the liposomes was determined by microfluidic resistive pulse sensing (MRPS) and freeze-fracture combined TEM. Small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC) and infrared spectroscopy (IR) were used to prove the unilamellarity, the ideal miscibility of the lipids and the ordered packing of the hydrocarbon chains of the lipids, respectively. Concentration of the lipids was determined by liquid chromatography–mass spectrometry (LC-MS). Results: The prepared liposomes proved to be unilamellar with narrow size distribution (83 nm avg.), as obtained by MRPS and TEM. DSC and IR measurements confirmed that the phospholipid bilayer of these liposomes is in the liquid-ordered phase, hence the area-per-lipid of 0.41 nm^2 was determined from WAXS measurements. Using the concentration of phospholipids from LC-MS measurements, the number concentration of liposomes was determined (8E+13 1/mL). Summary/conclusion: Liposomes containing saturated phospholipids are in the liquid-ordered phase, which can be utilized to determine the area-per-lipid using WAXS. This value, together with the independently determined size, and lipid concentration can be used to calculate the number concentration of liposomes. As the light scattering properties of liposomes matches that of EVs, liposome-based standards for optical measurements of EVs can be obtained with the presented techniques. Funding: This work was supported under grant numbers PD 121326 and NVKP_16-1-2016-0007 by NKFIH (Hungary). ZV was supported by the János Bolyai Research Fellowship. OWP1.12=LBF02.02 Plasma exosomes regulate proliferation and migration of vascular smooth muscle cells Kosuke Otani ^a, Mai Yokoya^b, Muneyoshi Okada^c and Hideyuki Yamawaki^b ^aLaboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Towada, Japan; ^bKitasato University, Towada, Japan; ^cKitasato University, School of Veterinary Medicine, Laboratory of Veterinary Pharmacology, Towada, Japan Introduction: We previously reported that systolic blood pressure in spontaneously hypertensive rats, an animal model of essential hypertension, was partly modulated by circulating exosomes (BBRC 2018). Vascular wall remodelling regulated by proliferation and migration of vascular smooth muscle cells (VSMCs) mediates development of hypertension. We aimed to clarify the effects of plasma exosomes derived from SHR and control Wistar Kyoto Rats (WKY) on proliferation and migration of VSMCs. Methods: Exosomes were isolated from rat plasma by an ultracentrifuge method, and identified through measurement of particle size distribution by a tunable resistance pulse sensing. For exploring exosome internalization in VSMCs, the isolated exosomes were labelled with PKH67 dye and observed by a fluorescence microscopy. Proliferation and migration of SMCs were determined by a bromodeoxyuridine incorporation and Boyden chamber assay, respectively. Actin cytoskeleton was visualized by a rhodamine-phalloidin staining. Expression of protein and microRNA in exosomes was determined by Western blotting and microarray, respectively. Results: There was no difference in size and concentration of plasma exosomes between WKY and SHR. Exosomes were incorporated into VSMCs, while the internalization of SHR exosomes was significantly lower than WKY exosomes. Both WKY and SHR exosomes similarly stimulated proliferation, migration and cytoskeletal changes such as formation of filopodia and lamellipodia in VSMCs. Heparin, an inhibitor of exosome internalization, completely blocked the migration and proliferation. Protein expression of CD9 and CD63, an exosomal marker, was significantly higher in exosomes from WKY than SHR. The expression of several microRNAs in SHR exosomes changed compared with WKY exosomes. Summary/conclusion: These results suggest that plasma exosomes play physiological, but not pathological, role on VSMCs irrespective of their origin (normotensive or hypertensive rats). Further research is required for determining whether the changes in molecular profiles of circulating exosomes mediate the development of high blood pressure in SHR. OWP1.13=LBF01.02 Colorectal cancer cell-derived exosome enhances microenvironmental angiogenesis through modulation of intracellular metabolism Atsushi Ikeda ^a, Satoshi Nagayama^b and Koji Ueda^c ^aCancer proteomics group, Cancer Precision Medicine Center, Japanese Foundation for Cancer Research, Tokyo, Japan; ^bDepartment of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan; ^cCancer Proteomics Group, Cancer Precision Medicine Center, Japanese Foundation for Cancer Research, Tokyo, Japan Introduction: For improvement of prognosis of colorectal cancer (CRC), detection at an earlier stage of CRC is essential. Exosomes are nanovesicles secreted from plasma membrane, and have potential to be served as biomarker carriers. In this study, we performed proteomic profiling of exosomes secreted from viable CRC tissues. Methods: To identify early detection biomarkers for CRC, we performed comprehensive proteome analysis of tissue-exudative extracellular vesicles (Te-EVs), which were obtained from culture media of freshly resected viable CRC tissue or adjacent normal mucosa (n = 17). Among the identified Te-EV proteins, we narrowed down the biomarker candidate by selecting proteins which are statistically upregulated (p < .05, fold change > 5.0) in Te-EVs from CRC tissues than those from adjacent normal tissues. Then we performed functional analysis of the biomarker candidate specifically. Results: Comprehensive LC/MS analysis identified 6,149 Te-EV proteins, in which 641 proteins showed significant upregulation in Te-EVs from CRC tissues (p < .05, fold change > 5. 0) compared to those from adjacent normal mucosa. We focused especially on GAM (p = 7.0 × 10–5, fold change = 7.4) as a novel biomarker candidate. GAM protein was significantly overexpressed in CRC tissues compared with adjacent normal mucosa. In EV-sandwich ELISA assay, the expression level of GAM on plasma EVs from CRC patients was significantly higher than that from healthy donors in EV-sandwich ELISA assay (n = 133, p = 4.0 × 10^–7). In addition, the uptake of GAM-overexpressing EVs enhanced vascular endothelial cell growth and angiogenesis through modulation of nitric oxide metabolism. Summary/conclusion: EV-GAM might have great potential as a target for both CRC diagnosis and therapy. Our strategy for identification of exosomal biomarker by proteomic profiling of Te-EV proteins can be applied to other cancers. OWP1.14=LBS02.01 Annexin V binding modulates the response of macrophages to mesenchymal stromal cell-derived extracellular vesicles Michele Grassi ^a, Michela Pozzobon^b, Melania Scarpa^c, Damiana Incendi^d, Alessia Giarraputo^e, Anna Maria Tolomeo^a, Marcin Jurga^f, Andrea Porzionato^g and Maurizio Muraca^h ^aDepartment of Women’s and Children’s Health – University of Padova, Padova, Italy; ^bUniversity of Padova, Padova, Italy; ^cIstituto Oncologico Veneto IOV-IRCCS, Padova, Italy; ^dUniversity di Padova Department DNS, Padova, Italy; ^eUniversity of Padova, Padova, Italy; ^fThe Cell Factory, Niel, Belgium; ^gDepartment of Neuroscience, University of Padua, Padova, Italy; ^hUniversity of Padova, Padova, Italy Introduction: We have previously shown that Annexin a5 (An5) binding to mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) enhances the anti-inflammatory properties of these nanoparticles in an animal model of colitis. However, the mechanisms underlying these effects are unknown. Here, we investigated the immunoregulatory effect of MSC-EVs with and without An5 binding on activated macrophages in vitro. Methods: Macrophages were isolated from mouse bone marrow and activated by INFgamma and LPS. Clinical grade Wharton Jelly-derived MSC-EVs were obtained from The Cell Factory (Esperite NV, Niel, Belgium) and quantified by Resistive Pulse Sensing analysis. 5,0E+05 macrophages were incubated with PBS (vehicle only, control, group 1) 5,0E+08 MSC-EVs (group 2), 5,0E+08 MSC-EVs added with 2 ug An5 (group 3) or with 2 ug free An5 (group 4). After 24 h, the cells were analysed by flow cytometry and RNA was extracted for RT-PCR analysis. Results: Incubation with MSC-EVs significantly increased only the expression of IL-10 in IFN-gamma/LPS-activated macrophages. Incubation with An5-MSC-EVs resulted in a significant induction in the expression of both pro- and anti-inflammatory cytokines, including TNF-alfa, IL-1Beta, IL-6, IL-10 and TGFbeta1. Incubation with free An5 induced only pro-inflammatory cytokines without affecting IL-10 and TGFbeta1 expression. The iNOS2/Arg1 ratio was reduced in both EV-treated groups, indicating a shift from M1 to M2 polarization. Summary/conclusion: In conclusion, both MSC-EVs and An5-MSC-EVs shift the macrophage phenotype from M1 to M2. The combined induction of TGFbeta1 and IL-10, observed only in An5-MSC-EV-stimulated macrophages, might be related to the immune-modulating characteristics of these modified EVs that contribute to the therapeutic effects observed in vivo. Funding: The BROAD MEDICAL RESEARCH PROGRAM AT CCFA supported this work OWP1.15=LBS03.01 Membrane-radiolabelled exosomes for comparative biodistribution analysis in immunocompetent and immunodeficient mice – A novel and universal approach Farid N. Faruqu^a, Julie Wang^a, Lizhou Xu^b, Luke McNickle^a, Ming-Yiu Chong^a, Mark Gurney^c, Aled Clayton^c, Lesley A. Smyth^d, Robert Hider^a, Jane Sosabowski^e and Khuloud Al-Jamal ^a ^aKing‘s College London, London, United Kingdom; ^bSchool of Cancer and Pharmaceutical Sciences, King‘s College London, London, United Kingdom; ^cCardiff University, Cardiff, United Kingdom; ^dUniversity of East London, London, United Kingdom; ^eQueen Mary University of London, London, United Kingdom Introduction: Exosomes have gained interest as novel drug nanocarriers due to their biological origin and role in intercellular biomolecule delivery. In-depth knowledge of their in vivo biodistribution is therefore essential. This work aimed to develop a reliable and universal method to radiolabel exosomes to study in vivo biodistribution in mice. Methods: Melanoma (B16F10 cells)-derived exosomes (ExoB16) were isolated and characterised for size, yield, purity, exosomal markers and morphology using Nanoparticle Tracking Analysis (NTA), protein measurements, flow cytometry and electron microscopy. Two radiolabelling approaches were explored – intraluminal labelling (111Indium entrapment via tropolone shuttling); and membrane labelling (111Indium chelation by covalently attached bifunctional chelator). Labelling efficiency and stability was assessed by gel filtration and thin layer chromatography. Melanoma-bearing immunocompetent (C57BL/6) and immunodeficient (NSG) mice were injected intravenously with radiolabelled ExoB16 (1x1011 particles) followed by metabolic cages study, whole body SPECT-CT imaging and ex vivo gamma counting at 1, 4 and 24 h post-injection. Results: Membrane-labelled ExoB16 (ML-ExoB16) showed superior radiolabelling efficiency and radiochemical stability compared to intraluminal-labelled ExoB16 (IL-ExoB16). Both IL- and ML-ExoB16 showed prominent accumulation in liver and spleen. IL-ExoB16 showed higher tumour accumulation than ML- ExoB16 (6.7% and 0.6% ID/g tissue, respectively), with the former showing similar value as its free tracer ([111]Trop). The superior stability of the membrane-labelling approach rendered its result more reliable and was used to compare ExoB16 biodistribution in melanoma-bearing immunocompromised (NSG) mice. Similar biodistribution profile was observed in both C57BL/6 and NSG mice, where prominent accumulation was seen in liver and spleen, apart from the lower tumour accumulation observed in the NSG mice. Summary/conclusion: Membrane radiolabelling of exosomes is a reliable approach that allows for both live imaging and quantitative biodistribution studies to be performed on potentially all exosome types without engineering parent cells. Oral with Poster Session 2 Chairs: Kazunari Akiyoshi; Muller FabbriLocation: Level B1, Lecture Room 13:30–15:00 OWP2.01=PS08.08 Identification of common EV markers in plasma using high-resolution flow cytometry Anders Askeland ^a, Jaco Botha^b, Rikke Wehner Rasmussen^b and Aase Handberg^b ^aAalborg University Hospital, Aalborg, Denmark; ^bDepartment of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark Introduction: Recent advancements in flow cytometry (FCM) have led to the development of high-resolution FCMs dedicated to the analysis of small particles (hFCM). hFCM studies have predominantly focused on the analysis of EVs expressing phosphatidylserine (PS). PS is enriched in microvesicles (MVs), wherein it is involved in lipid rearrangements responsible for MV budding. While PS also is expressed on exosomes, it is unknown whether it can be used as a universal marker for smaller EVs. In this study, we attempted to characterize proteins enriched in smaller EVs (CD9, CD63, CD81 and ADAM 10) and the relative co-expression of PS with each of these markers. Methods: FCM analysis was performed on an Apogee A60 Micro-PLUS. In brief, platelet-poor plasma (PPP) from healthy individuals was stained with lactadherin-FITC (PS+) and one of several EV surface markers enriched in smaller EVs. To evaluate the precise differences in PS and specific EV marker expression, the analysis was performed twice, (1) triggering on lactadherin and (2) each EV marker (CD9-PE, CD81-PE, CD63-PE, ADAM10-PE), separately. All antibodies were matched with appropriate isotope controls and centrifuged at 17,000g for 10 min prior to antibody labelling. EVs were defined as lactadherin or EV surface marker positive events ≤1000 nm. Results: Initial results indicate that CD9 is highly expressed on EVs and is not universally associated to PS. Triggering on PS revealed that 34.7% of all events were CD9 positive (CD9+|PS+). Conversely, triggering on CD9 resulted in a 2.1-fold increase in total events, where 17.0% of events were PS+ (CD9+|PS+). Inferring size from silica nanospheres, it appeared that populations containing CD9 (CD9+|PS+ and CD9+|PS-) were smaller (94.4–99.7% <180 nm) compared to populations that did not (PS+|CD9-; 85.6% <180 nm & 95.2% <300 nm). Interestingly, we did not detect CD81, CD63 or ADAM10 on EVs. We hypothesize that this is due to a low abundance of these markers in PPP from healthy individuals. Summary/Conclusion: Our findings demonstrate that hFCM can be used for the characterization of smaller EVs in PPP. Furthermore, we find that CD9+EVs do not universally express PS. From this point on, we plan to study enrichment of these EV phenotypes following a number of EV purification protocols, and determine whether EV isolation enable a more extensive characterization of smaller EVs. OWP2.02=PS08.09 Software to automate calibration and processing of flow cytometry data in clinical studies Edwin van der Pol ^s, Frank Coumans^b, Leonie de Rond^c, Aleksandra Gasecka^d, Najat Hajji^e, Rienk Nieuwland^b and Ton van Leeuwen^f ^aAmsterdam UMC, University of Amsterdam, Department of Biomedical Engineering and Physics, Amsterdam, Netherlands; ^bAmsterdam UMC, University of Amsterdam, Laboratory of Experimental Clinical Chemistry, Amsterdam, Netherlands; ^cAmsterdam University Medical Centers, Amsterdam, USA; ^d1st Chair and Department of Cardiology, Medical University of Warsaw, Warsaw, Poland; ^eAmsterdam University Medical Centers, Amsterdam, Netherlands; ^fdAmsterdam UMC, University of Amsterdam, Department of Biomedical Engineering and Physics, Amsterdam, Netherlands Introduction: In search of new biomarkers, flow cytometers are used in clinical studies to measure the concentration of specific extracellular vesicles (EVs). Flow cytometers measure light scattering and fluorescence of single EVs in a fluid stream. However, to realize data interpretation and comparison, light scattering and fluorescence signals and the flow rate require calibration. Moreover, flow cytometers generate large datasets. For example, a clinical study involving 60 patients, 30 controls, and 8 antibody labels covers 1224 data files, >33 gigabytes of data and >0.3 billion events. To manually calibrate and analyse such a dataset would take days if not weeks and is prone to human mistakes. Therefore, an urgent need exists for software to automate calibration and processing of flow cytometry data. Methods: We have developed software (MATLAB R2018a) to automatically process multiple .fcs files and (1) relate two scatter signals to the diameter in nm and refractive index (RI) of EVs, (2) express fluorescence signals in terms of molecules of equivalent soluble fluorochrome, (3) export calibrated channels to new .fcs files, (4) recognize unstable flow rates, (5) determine fluorescence thresholds, (6) apply gates, (7) create PDFs with scatter plots and (8) report statistics. We are using clinical studies to validate and apply the software. Results: Compared to manual thresholding, automatic thresholding results in a systematic decrease in counts of 10% and a maximum difference of 14% (n = 5). Using a high-end laptop, data processing takes typically a minute or several seconds per .fcs file with or without PDF reporting, respectively. Flow rate monitoring is useful for 61% of the data. The platelet marker CD61 stains 7% of the events with an RI >1.42, which are lipoproteins, and the concentration of these lipoproteins differed 4000-fold between individuals. Summary/Conclusion: We have developed software to automate calibration and processing of flow cytometry data in clinical studies, thereby reducing analyses time, preventing human mistakes and providing new insights. For example, non-specific labelling of antibodies to lipoproteins together with variations in lipoprotein concentrations emphasize the relevance of fasting before venipuncture. Our next step is to extend the software with machine learning. Funding: NWO-TTW VENI 15924. OWP2.03=PS08.10 Conventional, high-resolution and imaging flow cytometry: potentials, pitfalls and solutions for EV characterization Jaco Botha, Rikke Wehner Rasmussen, Mathilde Sanden and Aase Handberg Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark Introduction: Flow cytometry (FCM) has long been a preferred method for characterizing EVs, however their small size have limited the applicability of conventional FCM to some extent. Thus, high-resolution and imaging FCMs have been developed but not yet systematically evaluated. The aim of this presentation is to describe the applicability of high-resolution and imaging FCM in the context of EV characterization and the most significant pitfalls potentially influencing data interpretation. Methods: (1) First, we present a side-by-side comparison of three different cytometry platforms on characterising EVs from blood plasma regarding sensitivity, resolution and reproducibility: a conventional FCM, a high-resolution FCM and an imaging FCM. (2) Next, we demonstrate how different pitfalls can influence the interpretation of results on the different cytometry platforms. (3) Finally, we propose controls, solutions or workarounds for understanding and limiting the influence of each of these pitfalls. Results: (1) High-resolution FCM and imaging FCM displayed greater sensitivity and resolution compared to conventional FCM when measuring a mixture of nanospheres. Equally, both methods could detect larger concentrations of specific EV phenotypes than conventional FCM, where imaging FCM outperformed high-resolution FCM. Within day variability (n = 20 aliquots) was similar for conventional and high-resolution FCM, while imaging FCM had a markedly larger variability. Between day variability (n = 5 × 5 aliquots) was similar for all three platforms. (2) The three most substantial pitfalls variably influencing interpretation of results on the three platforms are non-specific binding of labels, antibody aggregates, and entities in the sample (i.e. lipoproteins) binding EV-defining dyes. (3) The most important strategies for circumventing these pitfalls are stringent matching, gating and comparison of antibodies and isotype controls, high-speed centrifugation of antibodies and labels prior to staining, and the use and interpretation of stained buffer controls and detergent treated samples. Summary/Conclusion: High-resolution and imaging FCM hold great potential for EV characterization. However, increased sensitivity also leads to new artefacts and pitfalls. The solutions proposed in this presentation provide useful strategies for circumventing these. OWP2.04=PS08.11 Convolutional neural networks for classification of tumour derived extracellular vesicles Wooje Lee ^a, Aufried Lenferink^a, Cees Otto^b and Herman Offerhaus^a ^aUniversity of Twente, Enschede, Netherlands; ^bMedical Cell Biophysics, University of Twente, Enschede, Netherlands Introduction: Raman spectroscopy probes molecular vibration and thus reveals chemical information of a sample without labelling. This optical technique can be used to study the chemical composition of diverse extracellular vesicles (EVs) subtypes. EVs have a complex chemical structure and heterogeneous nature so that we need a smart way to analyse/classify the obtained Raman spectra. Machine learning (ML) can be a solution for this problem. ML is a widely used strategy in the field of computer vision. It is used for recognizing patterns and images as well as classifying data. In this research, we applied ML to classify the EVs’ Raman spectra. Methods: With Raman optical tweezers, we obtained Raman spectra from four EV subtypes – red blood cell, platelet PC3 and LNCaP – derived EVs. To classify them by their origin, we used a convolutional neural network (CNN). We adapted the CNN to one-dimensional spectral data for this application. The ML algorithm is a data hungry model. The model requires a lot of training data for accurate prediction. To further increase our substantial dataset, we performed data augmentation by adding randomly generated Gaussian white noise. The model has three convolutional layers and fully connected layers with five hidden layers. The Leaky rectified linear unit and the hyperbolic tangent are used as activation functions for the convolutional layer and fully connected layer, respectively. Results: In previous research, we classified EV Raman spectra using principal component analysis (PCA). PCA was not able to classify raw Raman data, but it can classify preprocessed data. CNN can classify both raw and preprocessed data with an accuracy of 93% or higher. It allows to skip the data preprocessing and avoids artefacts and (unintentional) data biasing by data processing. Summary/Conclusion: We performed Raman experiments on four different EV subtypes. Because of its complexity, we applied a ML technique to classify EV spectra by their cellular origin. As a result of this approach, we were able to classify EVs by cellular origin with a classification accuracy of 93%. Funding: This work is part of the research programme [Cancer-ID] with project number [14197] which is financed by the Netherlands Organization for Scientific Research (NWO). OWP2.05=PS08.12 Microfluidic electrochemical aptasensor for detection of breast cancer-derived exosomes in biofluids Leila Kashefi-Kheyrabadi, Sudesna Chakravarty, Junmoo Kim, Kyung-A Hyun, Seung-Il Kim and Hyo-Il Jung Yonsei University, Seoul, Republic of Korea Introduction: Exosomes are nano-sized extracellular vesicles, which are emerging as potential noninvasive biomarkers for early diagnosis of cancer. However, the small size and heterogeneity of the exosomes remain significant challenges to their quantification in the biofluids. In the present research, a microfluidic electrochemical biosensing system (MEBS) is introduced to detect ultra-low levels of breast cancer cell-derived exosomes (BCE). Methods: Fabrication procedure of MEBS comprises three main steps: first, biosensing surface was prepared by immobilizing EPCAM binding aptamer (EBA) on a nanostructured carbon electrode. The nanostructured surface (NS) consists of 2-D nanomaterials including MoS2 nano-sheets, graphene nano-platelets, and a well-ordered layer of electrodeposited gold nanoparticles. The NS was well characterized with FESEM and EDX. FESEM analysis showed a well-ordered gold nano-structuring for 50 nM of gold solution. Furthermore, EDAX analysis confirmed >60% coverage of gold nanoparticles on NS compared to bare carbon electrode. At the second step, a herringbone structured microfluidic channel, which is able to enrich BCE was designed and fabricated. Finally, microfluidic channel was integrated to biosensing surface. Different concentrations of exosome solutions was introduced and enriched to biosensing surface (SPCE/NS/GNP/EBA) using microchannel. After capturing BCEs on the sensing surface a secondary aptamer labelled with silver nanoparticles (SNPs) as redox reporter was introduced to the sensing surface. Results: Direct electro-oxidation of SNPs was monitored as analytical signal. The unique design of microchannel in combining with high specific interaction between BCE and EBA provided a high sensitive detection of BCE as low as ~100 exosomes/μL. Summary/Conclusion: The unique design of MEBS provides a highly sensitive accurate platform for detection of ultra-low levels of cancer-derived exosomes. This tool holds great potential for early cancer diagnosis in clinical applications. OWP2.06=PS08.13 A software suite allowing standardized analysis and reporting of fluorescent and scatter measurements from flow cytometers Joshua Welsh and Jennifer C. Jones Translational Nanobiology Section, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, USA Introduction: Single vesicle analysis using flow cytometry is an extremely powerful technique to allow identification of unique proteins in biological samples, as well as enumerating the changes in concentrations. While small particle analysis (for viruses and large microparticles) using flow cytometry has been conducted for several decades, there is no comprehensive method for standardization of such studies. Therefore, we developed a suite of flow cytometry post-acquisition analysis software (FCM[PASS]) tools that enable the conversion of scatter and fluorescent axes to standardized units using appropriate controls, writing standardized units to .fcs files for sharing upon publication with open repositories, and exporting templates of obtained data. Methods: Standalone software packages for scatter and fluorescent standardization were built using MATLAB. The scatter software is based upon Mie modelling and is capable of predicting the optical collection angle of the instrumentation and reporting the Mie modelling criteria in a standardized way, making it possible to reproduce the models and flow cytometry settings. Fluorescent standardization data uses least-squares linear regression to enable conversions of arbitrary unit scales to molecules of equivalent soluble fluorophore (MESF) using MESF calibration beads. Results: The FCM[PASS] software converts arbitrary fluorescence units to MESF units and writes them to data files for clearer reporting and sharing of data. FCM[PASS] also converts arbitrary scatter units to a measurement of scattering cross-section using modelling software that predicts the collection angle of the instruments and normalizes the data automatically. Summary/Conclusion: Utilization of our FCM[PASS] software can help the EV flow cytometry more easily implement standardization into their experimental analysis and the use of the output templates can make reporting more consistent. While currently available MESF controls can be further optimized for small particles, we believe their utilization along with the other controls, can bring a new era to the reporting of EV research using flow cytometry. This will be particularly useful for future comparison and validation of translational studies and will enable better understanding and utilization of EVs across a broad range of disciplines. OWP2.07=PF05.08 Biogenesis of JC polyomavirus associated extracellular vesicles depends on neutral sphingomyelinase 2 Jenna Morris-Love ^a, Bethany O’Hara^b, Gretchen Gee^a, Aisling Dugan^b, Benedetta Assetta^c, Sheila Haley^a and Walter Atwood^a ^aBrown University, Providence, USA; ^bAssumption College, Worcester, USA; ^cBrown Univerisity, Providence, USA Introduction: JC polyomavirus is a non-enveloped virus that causes progressive multifocal leukoencephalopathy (PML) in immunocompromised patients. JCPyV infects cells by first binding to the major attachment receptor lactoseries tetrasaccharide C (LSTc), followed by the serotonin receptor 5-hydroxytryptamine type 2 required for entry. In PML, JCPyV undergoes lytic infection in oligodendrocytes and astrocytes, both of which have been shown to lack LSTc. Further, deep sequencing has shown that viral quasispecies existing in PML patients contain mutations in the sialic acid binding pocket of the major viral capsid protein, rendering these virions incapable of binding LSTc. We have recently demonstrated that JCPyV is packaged into extracellular vesicles (EVs) that can spread the virus, potentially overcoming this paradox. Here, we begin to characterize the biogenesis of this EV-virus association by examining endosomal sorting complexes required for transport (ESCRT) proteins and neutral sphingomyelinase 2 (nSMase2). Methods: Cambinol was used to specifically target nSMase2 activity. Knockdown cell lines were created with shRNA targeted against ALIX, TSG101 or SMPD3. SMPD3 was also targeted using CRISPR/Cas9 genetic knockout in separate cell lines. Knockdown was confirmed by qPCR and/or Western blot, and knockout by next generation sequencing. EV were concentrated by differential centrifugation and evaluated by transmission electron microscopy, Western blot, nanoparticle tracking analysis, infection and qPCR for protected viral genomes. Infection was scored by immunofluorescence analysis with antibodies against the major viral capsid protein VP1. Results: We found that depletion of nSMase2 by cambinol, genetic knockdown or knockout caused a reduction in spread of JCPyV over time. Knockdown and knockout SMPD3 cell lines produced less infectious EV. In the absence of nSMase2, cells produced more EV but there were fewer protected genomes associated with the EV. Knockdown of Alix or TSG101 had no effect on the infectivity of EV or the production of EV. Summary/Conclusion: Overall, our studies found that biogenesis of JCPyV associated EVs depends upon the enzymatic activity of nSMase2 and not the ESCRT-related proteins Alix or TSG101. Funding: NIH R01NS043097. OWP2.08=PF05.09 Exosomes mediate the antiviral activity of interferon-β against Zika virus infection Shuang Li, Shilin Li and Limin Chen Provincial Key Laboratory for Transfusion-Transmitted Infectious Diseases, Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China (People’s Republic) Introduction: IFNβ-induced exosomes (Exo-IFNβ) may impact on viral dissemination or antiviral immunity and therefore involve in the pathogenesis of many infectious pathogens. However, little is known about its underlying mechanisms. To better understand how Exo-IFNβ perform its antiviral effect, we employed RNA sequencing analysis to explore the exosomal expression profiles of lncRNA and mRNA related to viral infections. We hypothesized that exosomes can regulate viral infection through transmitting enclosed specific lncRNAs into neighbouring cells to inhibit viral replication. Methods: Exosomes were purified from A549 with/without IFNβ treatment by serial centrifugation followed by sucrose density gradient purification, and characterized by TEM and Western Blot. ELISA assay were performed on purified exosome fractions to demonstrate that they are free of IFNβ. Zika virus (ZIKV) replication was assayed by real-time PCR. Results: ZIKV replication was significantly suppressed in A549 cells pretreated with Exo-IFNβ followed by ZIKV infection. Moreover, we found that anti-ZIKV effect of Exo-IFNβ is IFN-independent because ZIKV replication was also decreased in U5A cells (IFN-α/β receptor IFNAR deficient) pre-treated with Exo-IFNβ. Similar results were observed in Dengue virus and HCV infections. RNA sequencing analysis found several lncRNAs and mRNAs were differentially expressed and function annotation and pathway analysis demonstrated that the differentially expressed genes were involved in many functions and pathways, including antiviral infection. To validate the RNA sequencing analysis results, some lncRNAs were selected to test their expression levels by qPCR. We are in the process of deciphering the mechanism employed by these exosomal lncRNAs in antiviral activity independent of inteferon. Summary/Conclusion: We believe that understanding the antiviral functional molecules wrapped in exosomes may help design exosomes as efficient vehicles for antiviral therapy. Funding: Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2016-12M-3-025) OWP2.09=PS02.09 Deciphering the role of extracellular vesicles on the blood–brain barrier during Zika virus infection Antonios Fikatas, Sam Noppen, Peter Vervaeke, Jordi Doijen, Mohammed Benkheil, Christophe Pannecouque and Dominique Schols Laboratory of Virology and Chemotherapy, Rega Institute, KU Leuven, Belgium, Belgium Introduction: The association of Zika virus (ZIKV) with severe neurological disorders has gained increased interest over the last decade. However, the mechanism by which ZIKV crosses the blood–brain barrier (BBB) and reaches the brain remains to be elucidated. It is known that viruses incorporate viral material in extracellular vesicles (EVs) as a spreading strategy. These membrane-enclosed vesicles play a vital role in intercellular communication. Currently, there is a lack of knowledge on the possible involvement of EVs in ZIKV pathogenesis. Our study aims to unravel the role of EVs in ZIKV RNA transmission to the brain, via the BBB. Methods: Human brain microvascular endothelial cells (HBMEC/D3) were used in our study since they represent the BBB in vitro. Three different EV isolation methods (precipitation kit, density gradient and size exclusion chromatography combined with the density gradient) were performed. Western blot, Transmission electron microscopy and Nanosight tracking analysis confirmed the presence of EVs in the supernatant of HBMEC/D3 cells. The presence of ZIKV RNA in infected-EVs (IEVs) was evaluated by immunofluorescence and qPCR. In addition, the effect of IEVs on the BBB was assessed using a label-free impedance-based biosensor (ECIS, Applied BioPhysics). Results: We confirmed the presence of viral components in our IEVs, including the NS1 and E proteins of ZIKV. The obtained IEVs were able to reinfect susceptible cells, even after being pretreated with RNase A. This indicates that the viral RNA resides inside the IEVs. Using impedance measurements on HBMEC/D3 cell monolayers, we observed that IEVs, as well as virus control caused similar and temporal disturbances on the monolayer’s integrity within 30 min post infection. No disturbances were seen upon addition of non-infected EVs. Summary/Conclusion: Our study demonstrates that EVs-derived from ZIKV-infected cells are able to transfer proteins and viral RNA to recipient cells. Since both IEVs and viral particles can induce similar changes on barrier’s integrity it is possible that IEVs are involved in an alternative mechanism of ZIKV transmission. OWP2.10=PF12.10 HIV-specific antibody mediated targeting of ENV+ tissues by exosomes Zou Xue, Yuan M’eng, Zheng Nan and Wu Zhiwei Nanjing University, Nanjing, China (People’s Republic) Introduction: Antiretroviral therapy can effectively suppress HIV replication in the peripheral blood to an undetectable level. However, efforts to eradicate the latent virus in reservoirs remain a challenge and are a major obstacle in the treatment of HIV patients. Exosomes exhibit huge promise as an endogenous drug delivery nanosystem for delivering drugs to reservoir tissues given their unique properties, including low immunogenicity, innate stability, high delivery efficiency and mostly importantly the ability to penetrate solid tissues due to their lipophilic properties. Methods: In this study, we engineered and expressed the ScFv of a high affinity HIV-specific monoclonal antibody, 10E8, on exosome surface. Exosomes from 293T cells were loaded with curcumin via saponin, with efficient up to 34%. 10E8ScFv-expressing exosomes (10E8-Exo) showed highly efficient targeting of and curcumin delivery to CHO cell that expresses a trimeric gp140 on its surface (ENV+ cells) in vitro as demonstrated by confocal imaging and flow cytometry. We showed that 10E8-Exo could effectively bind to CHO cell that expresses a trimeric gp140 on its surface. The exosomes loaded with curcumin, a chemical that was shown to kill HIV-infected cells, showed specific killing of the trimeric gp140-expressing CHO cells. In an NCG mouse model that was grafted with the tumorigenic gp140-CHO cells and developed solid tissue tumours intravenously injected 10E8-Exo targeted the ENV-expressing tissues and delivered curcumin to induce a strong suppression of the ENV+ tumour growth with a low toxicity. Results: Our results demonstrated that engineered exosomes can deliver anti-HIV agents to solid tissues by specifically targeting cells expressing viral env and induce cell killings. Summary/Conclusion: It suggesting that such an approach can be developed for eradicating virus-infected cells in tissue reservoir. Funding: This study was supported by The National Key Research and Development Program of China (2016YFC1201000), Nature Science Foundation of Jiangsu Province (BY2015069-02) and National Nature Science Foundation of China (81672020). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. OWP2.11=PS02.10 In vivo testing of OMV-based vaccine prototypes against Gallibacterium anatis Fabio Antenucci ^a, Homa Arak^b, Jianyang Gao^b, Toloe Allahghadry^b, Ida Thøfner^b and Anders Miki Bojesen^c ^aUniversity of Copenhagen, København S, Denmark; ^bUniversity of Copenhagen, Copenhagen, Denmark; ^cUniversity of Copenhagen, Copenhagen, USA Introduction: Outer membrane vesicles (OMVs) are produced by the majority of Gram-negative bacteria. Thanks to the antigenic similarity between OMVs and the bacterial outer membrane, OMVs have proven to be promising for the development of novel vaccines against bacterial pathogens. In this work, we describe the testing of OMV-based vaccine prototypes against Gallibacterium anatis, a Gram-negative pathogen of great veterinary interest. Methods: OMVs were isolated from a G. anatis hypervesiculating mutant using a modified version of the Hydrostatic Filtration protocol described by Musante et al. (2014). 120 16-week-old Lohmann-Brown chickens were divided in six groups and immunized twice intramuscularly with different combinations of buffer (controls), OMVs and selected recombinant immunogens. Two weeks after second immunization, the effectiveness of the immunization regimes adopted was tested by challenging the animals intraperitoneally with live CFUs from a heterologous G. anatis strain. One week post-challenge, the animals were sacrificed and an established lesion score model was used during necropsy to evaluate the clinical outcome of infection. Results: Statistical analysis of the recorded lesion scores showed that the group immunized with G. anatis OMVs presented an average total score of 2.95, as opposed to an average total score of 8.77 in the control group. The approximately three-fold reduction in total average lesion score observed demonstrates that immunization with G. anatis OMVs is able to effectively decrease the morbidity of G. anatis infection in the immunized animals. Summary/Conclusion: Our results show that G. anatis OMVs represent a promising candidate for the development of cost-effective vaccination strategies for the prevention of G. anatis infections in a cross-serovar manner. Accordingly, we hypothesize that dose/response optimization and the enrichment of G. anatis OMVs with selected immunogens should result in an improvement of the effectiveness of the vaccination regime proposed. Funding: This research project is being funded by a grant from Huvepharma (https://www.huvepharma.com/). OWP2.12=PT05.04 Identification of a protein that presumably controls bacterial vesiculation in response to the extracellular environments Fumiaki Yokoyama ^a, Jun Kawamoto^a, Chen Chen^a, Tomoya Imai^b and Tatsuo Kurihara^a ^aInstitute for Chemical Research, Kyoto University, Uji, Japan; ^bResearch Institute for Sustainable Humanosphere, Kyoto University, Uji, Japan Introduction: Many bacteria utilize extracellular membrane vesicles (EMVs) for survival in their growing environments through communication with others, pathogenesis, and biofilm formation. Therefore, the amounts and the components of EMVs should be tuned in response to the conditions. Although several vesiculation mechanisms are suggested, little is known how bacteria control vesiculation in response to the environments. A bacterium Shewanella sp. HM13 has nine fold higher lipid-secretion capability in EMV fractions than Escherichia coli, and its EMVs contain a major protein (P49), which is not required for vesicle production. We used mutant EMVs that lack P49 to identify minor components of EMVs that may control vesiculation. Methods: EMVs were subjected to 2D gel-based proteomics by peptide mass fingerprinting. Within the identified proteins, the function of a sensor protein homolog, HM1275, was analysed by swarming assay and lipid-staining to quantify EMVs produced in various media. Changes in the number of EMVs depending on culture media were quantified by tunable resistive pulse sensing method. Results: A protein with a PAS domain and a methyl-accepting chemotaxis protein (MCP) sensing domain, HM1275, was identified in the EMVs. Although some MCPs are related to flagellar motility by binding some attractants, the flagellar motility of Delta-hm1275 was not significantly different from that of WT. Although the amounts of EMVs produced by WT were increased in response to the concentration of casamino acids in poor nutrient medium, those by Delta-hm1275 were not. Summary/Conclusion: A putative sensor protein, HM1275, was identified in EMVs and may recognize the extracellular environments by binding signal molecules in casamino acids to control vesiculation. Although further studies are required to reveal the signals and the sensing pathways, the results obtained in this study indicate that bacterial vesiculation is controlled by extracellular environments, and artificial control of vesiculation with extracellular signals would be useful in applications such as suppression of vesicle-dependent pathogenicity. Funding: Japan Society for Promotion of Science Research Fellowship for Young Scientists OWP2.13=PT05.05 Prokaryotic BAR domain-like protein BdpA promotes outer membrane extensions Daniel A. Phillips ^a, Lori Zacharoff^b, Cheri Hampton^c, Grace Chong^b, Brian Eddie^d, Anthony Malanoski^d, Shuai Xu^b, Lauren Ann Metskas^e, Lina Bird^f, Grant Jensen^e, Lawrence Drummy^c, Moh El-Naggar^b and Sarah Glaven^d ^aAmerican Society for Engineering Education – U.S. Naval Research Laboratory, Washington, USA; ^bUniversity of Southern California, Los Angeles, USA; ^cMaterials and Manufacturing Directorate, Air Force Research Laboratory, Dayton, USA; ^dU.S. Naval Research Laboratory, Washington, USA; ^eCalifornia Institute of Technology, Pasadena, USA; ^fNational Research Council, Washington, USA Introduction: Bin/Amphiphysin/RVS (BAR) domains belong to a superfamily of membrane-associated coiled-coil proteins that influence membrane curvature. BAR domains are ubiquitous in eukaryotes and associated with membrane curvature formation, vesicle biogenesis/trafficking, protein scaffolding and intracellular signalling. While advances in protein domain prediction have facilitated the identification of several BAR domain proteins, they have yet to be characterized in bacteria. Here we identified a putative BAR domain-containing protein enriched in the outer membrane vesicles (OMVs) of Shewanella oneidensis MR-1, a dissimilatory metal-reducing bacteria known to produce outer membrane extensions (OMEs) that are suspected to facilitate long distance extracellular electron transfer (EET) but whose physiological relevance and mechanism of formation remain unknown. Methods: Purified S. oneidensis OMVs were prepared by filtration and ultracentrifugation for comparative proteomics with cell-associated outer membrane proteins or for electrochemical measurements. Protein domains were predicted using HMMSCAN and CDD-search. OME formation and phenotype analyses were performed in situ by confocal and cryo-electron microscopy. Results: The putative BAR domain-like protein BdpA was highly enriched in OMVs compared to cell-associated outer membranes. During OME biogenesis, WT S. oneidensis OMEs progress from elongated vesicle chains to narrow, tubule-like extensions while ΔbdpA OMEs remain as disordered vesicle chains. Purified OMVs from these strains are electrochemically active, with redox signals consistent with multiheme outer membrane cytochromes, supporting the role of OMEs in EET. Heterologous BdpA expression promotes OME formation in Marinobacter atlanticus and Escherichia coli, suggesting BdpA membrane sculpting activity is inducible and transferrable. Summary/Conclusion: The ability of BdpA to promote OME formation and maturation into tubules in vivo supports BdpA as a comparator for BAR domain protein activity in bacteria. Funding: US DoD Synthetic Biology for Military Environments (SBME) Applied Research for the Advancement of Science and Technology Priorities (ARAP) NSF Dimensions: DEB-1542527 US DOE: DE-FG02-13ER16415 OWP2.14=PF07.10 Isolation of extracellular vesicles from extracellular matrix based hydrogel 3D cell cultures Jens Luoto ^a, Lea Sistonen^b and Eva Henriksson^b ^aCell Biology, Biosciences, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland; ^bTurku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland Introduction: Cancer-derived extracellular vesicles (EVs) are commonly studied and isolated from two-dimensional (2D) cell cultures. Nevertheless, three-dimensional (3D) culture systems with extracellular matrix (ECM) provide physiologically more relevant system to mimic in vivo tumour growth and progression of invasion. However, there are currently no methods to efficiently isolate EVs from ECM-based 3D cultures. For that purpose, we established a protocol for isolating EVs from cancer cells growing in a 3D ECM-based hydrogel. Methods: Human prostate cancer PC3 cells were grown in 3D to form spheroids in a commercially available ECM-based hydrogel and the growth media was collected every two days for a period of 14 days, during which the spheroids grew invasive. The respective media were differentially centrifuged at 2, 10 and 100 Kg and the pellets were resuspended in PBS. The EVs were analysed by western blotting (WB) against the common EV markers CD81, CD63 and CD9. Results: Our preliminary data shows a step-wise increase of the EV markers in the media as the PC3 spheroids formed, expanded and invaded to the surrounding 3D ECM. The EVs produced by non-invasive or invasive spheroids are currently being characterized with nano tracking analysis, electron microscopy and WB. Summary/Conclusion: This study demonstrates that EVs can be isolated from 3D ECM-based hydrogel cell cultures, which recapitulate the tissue architecture of solid tumours. Our results suggest that 3D cancer cell cultures have dynamic EV secretion determined by the phenotype of the spheroids. Taken together, we present a novel protocol for EV isolation from a 3D culture system and provide a platform to investigate EVs from in vivo mimicking conditions. Funding: This project is funded by Magnus Ehrnrooth Foundation, K. Albin Johansson Foundation and Åbo Akademi University. OWP2.15=PT07.07 Diagnostic microRNA biomarkers from circulating extracellular vesicles for early detection of pneumonia and severe secondary complications Stefanie Hermann ^a, Benedikt Kirchner^a, Dominik Buschmann^b, Melanie Märte^c, Florian Brandes^c, Stefan Kotschote^d, Michael Bonin^e, Marlene Reithmair^f, Matthias Klein^g, Gustav Schelling^c and Michael Pfaffl^h ^aDivision of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Germany; ^bTUM School of Life Sciences Weihenstephan, Division of Animal Physiology and Immunology, Freising, Germany; ^cDepartment of Anesthesiology, University Hospital, Ludwig-Maximilians-University Munich, München, Germany; ^dIMGM Laboratories GmbH, Planegg, Germany; ^eIMGM Laboratories GmbH, Planegg, Germany, Martinsried, USA; ^fInstitute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, München, Germany; ^gDepartment of Neurology, University Hospital, Ludwig-Maximilians-University Munich, München, Germany; ^hAnimal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany Introduction: Pneumonia remains one of the most deadly communicable diseases, causing three million deaths worldwide in 2016. Extracellular vesicles (EVs) are pivotal during signal transfer in the pathogenesis of inflammatory lung diseases. Since identifying pneumonia is particularly challenging in high risk groups (e.g. the elderly or infants), which often present with atypical symptoms and are at high risk for secondary complications such as sepsis or acute respiratory distress syndrom (ARDS), new approaches for early diagnosis are required. In this study we identified EV microRNAs (miRNAs) as potential biomarkers for inflammatory changes of the pulmonary tissue. Methods: Our study included 13 patients with community-acquired pneumonia, 14 ARDS patients, 22 patients with sepsis and 31 healthy controls. After precipitating EVs from 1 mL serum, total RNA was extracted. Subsequent to library preparation and small RNA-Seq, differential gene expression analysis was performed using DESeq2. Data were filtered by mean miRNA expression of ≥50 reads, minimum twofold up or down regulation and adjusted p-value ≤0.05. Results: The mean relative miRNA frequency varied slightly between the different groups and was highest in volunteers. Short sequences (<16 nucleotides), probably degradation products from longer coding and non-coding RNA species, were predominantly detected in patients. Based on unsupervised clustering, patients could be distinctly separated from healthy individuals. Although 21 miRNAs were significantly regulated in all patient groups compared to healthy controls, different disorders showed unique miRNA expression profiles. Distinct miRNA subsets were identified, which are applicable to indicate disease progression from limited inflammation present in pneumonia to severe inflammatory changes as seen in ARDS and sepsis. Summary/Conclusion: This study shows that EV miRNA biomarkers have potential for diagnosis of pneumonia and to indicate disease progression towards severe lung injury. Our findings are of clinical relevance, as the timely diagnosis of pneumonia can be challenging, and secondary complications such as ARDS and sepsis might be prevented by early intervention and treatment. Funding: This study was supported by the German Federal Ministry for Economic Affairs and Energy under the programme “Zentrales Innovationsprogramm Mittelstand”. Oral with Poster Session 3 Chairs: Michael Pfaffl; Ryuichi OnoLocation: Level B1, Hall A 13:30–14:15 OWP3.01=LBT02.02 Using plasma to identify neural biomarker for antidepressant response in a treatment resistant cohort Corina Nagy ^a, Saumeh Saeedi^b, Jean-Francois Theroux^c, Marina Wakid^c, Naguib Mechawar^c and Gustavo Turecki^b ^aDHRC- McGill University, Verdun, Canada; ^bMcGill University, Verdun, Canada; ^cMcGill University, Verdun, Canada Introduction: Small extracellular vesicles (SEV) have emerged as candidate biomarkers in many complex diseases. An important characteristic of SEVs is their ability to bidirectionally cross the blood-brain barrier. This is particularly important in the context of major depressive disorder (MDD), where biomarkers are obtained from peripheral tissue and have been hard to relate to changes in brain functioning. 60% of MDD patients do not respond to their first antidepressant drug therapy (ADT) and treatment options are entirely at the discretion of the physician. Findings that can predict ADT response as well as provide insight into central mechanistic changes could revolutionize MDD treatment. The aim of this study is to profile exosomal microRNA (miRNA) in the context of ADT response in people with treatment-resistant depression. miRNA can act as biomarkers and might influence recipient cells to provide insight on disease-relevant mechanistic changes. Methods: This pilot uses plasma from 10 controls and 10 patients with MDD (5 ADT responders (RES), and 5 non-responders (NRES)) from baseline (T0, before treatment). SEVs were isolated using a size exclusion column from Izon Science (Christchurch, New Zealand). Each isolation was divided into a “whole exosome” fraction and an immunoprecipitated “(NDE)” fraction using neural marker L1CAM. Quantitation and size determination was done using Tunable Resistive Pulse Sensing (TRPS) on the qNano gold. RNA was also extracted from SEVs from both fractions. The 4N-small RNA-Seq (Galas) protocol was used for library preparation. Results: We found that the range of SEVs in the NDE fraction was smaller than the pool of all exosomes combined. Further, SEVs from all depressed patients were significantly smaller than controls irrespective of the fractions. Our sequencing results showed an increase of miR-151a-3p and miR-3168 in NRES, and miR-22-3p in RES. These results were specific to the NDE fraction. Summary/conclusion: We have identified three potential biomarkers for ADT response which are uniquely present in the neural-derived fraction of peripheral SEVs. Funding: Canadian Institutes of Health Research OWP3.02=PT09.13 Immunocapturing of tumour-derived extracellular vesicles on micropatterned and antibody-conjugated surfaces for individual correlative light, probe and electron measurements Pepijn Beekman ^a, Agustin Enciso-Martinez^b, Cees Otto^b and Séverine Le Gac^c ^aWageningen University, Wageningen, Netherlands; ^bMedical Cell Biophysics, University of Twente, Enschede, Netherlands; ^cApplied Microfluidics for BioEngineering Research, University of Twente, The Netherlands, Enschede, Netherlands Introduction: Tumor-derived extracellular vesicules (tdEVs) are promising biomarkers for cancer patient management. The screening of blood samples for tdEVs shows prognostic power comparable to screening of tumour cells. However, due to the overlap in size between tdEVs, non-cancer EVs, lipoproteins and cell debris, new approaches, not only based on size, are required for the reliable isolation of tdEVs and their quantification. We report an integrated analysis methodology to study single tdEVs using correlative data from scanning electron microscopy (SEM), Raman imaging and atomic force microscopy (AFM) to obtain a comprehensive dataset allowing identifying features unique to tdEVs. Methods: Indium tin oxide (ITO)-coated fused silica was selected for its low Raman background. Substrates (1 × 1 cm^2) featuring position-dependent markings (“navigation marks”) patterned by photolithography were modified with a monolayer of amino dodecyl phosphonic acid. The amine moieties were next reacted with poly(ethylene glycol) diglycidyl ether, forming an anti-biofouling layer. Anti-EpCAM antibodies were subsequently covalently bound on this surface. Samples of both tdEVs obtained from LNCaP cell lines and RBC-derived EVs were then introduced to the surfaces. Finally, non-specifically bound EVs were washed away before SEM, AFM and Raman measurements were performed. Results: Multiple objects were captured on the fully functionalized ITO surfaces, according to SEM imaging, while in negative control experiments (lacking functionalization or lacking antibody or using EpCAM-negative EVs), no object was detected. Principal component analysis of their Raman spectra, previously demonstrated to be able to distinguish tdEVs from RBC-derived EVs, revealed the presence of characteristic lipid bands (e.g. 2851 cm^−1) in the captured tdEVs. AFM showed a surface coverage of ∽4 × 10^5 EVs per mm^2 with a size distribution similar to that found by NTA. Summary/Conclusion: A platform was developed for multi-modal analysis of selectively isolated tdEVs for their multimodal analysis. In the future, the scope of this platform will be extended to other combinations of probe, light and electron microscopy techniques to relate additional parameters describing the captured EVs. Funding: Funded by NWO Perspectief. OWP3.03=PT09.14 The development of a scalable extracellular vesicle subset characterization pipeline Joshua Welsh ^a, Julia Kepley^b and Jennifer C. Jones^a ^aTranslational Nanobiology Section, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, USA; ^bTranslational Nanobiology Lab, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, USA Introduction: Liquid biopsies offer an important alternative to tumour biopsies that may be limited by the challenges of invasive procedures. We hypothesize that circulating Extracellular Vesicles (EVs) and their cargo may provide a useful surrogate biopsy method. Due to their small diameter (30–1000 nm), EVs migrate from the tissue into the peripheral circulation and provide a snapshot of the producing cells. Our lab has developed a first-in-class pipeline to use single cell – omics methods to characterize EV heterogeneity with high-sensitivity by combining multiplex assays and our custom MultiPlex Analysis post-acquisition analysis software (MPA[PASS]), with subsequent high-resolution, single EV flow cytometric (FCM) methods. Methods: A stan-dalone software package was developed in MATLAB to allow importation of multiplex flow cytometry output data. The package enables data quality screening of detection antibodies, bead recovery and data normalization methods. The software is equipped to handle large data sets comprising hundreds/thousands of phenotypes and samples. Data can be visualized in a variety of ways along with clustering using multidimensional data analysis techniques. All software outputs can be exported in a standardized templates containing metadata for reporting, as well as uploaded into atlases such as Genboree, where multiplex data can be stratified by RNAseq datasets. Analysis using this pipeline has been conducted using human samples from a variety of mediums including CSF, serum and plasma comparing EV phenotypes. Results: Our multiplex approach and MPA[PASS] software allows the use of single cell -omics tools for EV subset analysis in a manner that will elucidate the biological significance and function of different types of EVs. This high-throughput pipeline evaluates hundreds of EV protein profiles and will allow evaluation of millions of RNA:protein profiles in an unprecedented manner. Integration of RNA sequencing with protein characterization could provide an entirely new way of understanding EV regulation and function. Summary/Conclusion: Our data show this form of EV profiling provides a way to monitor clinical responses early in the course of treatment, which may ultimately improve patient care and outcomes. OWP3.04=PS04.13 An integrated microfluidic device for selective exosome isolation from human plasma Hogyeong Gwak^a, Junmoo Kim^b, Leila Kashefi-Kheyrabadi^b, Seung-Il Kim^b, Kyung-A Hyun ^b and Hyo-Il Jung^b ^aSchool of Mechanical Engineering, Yonsei University, Seoul, Republic of Korea; ^bYonsei University, Seoul, Republic of Korea Introduction: Extracellular vesicles released by many cell types circulate in blood vessel and play a key role in intercellular communication. Exosomes are 30–150 nm membrane vesicles and are also shed by both normal and cancer cells. Cancer cells are known as very heterogeneous, so exosomes are also heterogeneous and have different surface expression markers. Cancer-derived exosomes contain unique cargo determined by the molecular characteristics of cancer cells. Therefore, it is very important to selectively separate exosomes depending on surface expression for downstream analysis. We designed an integrated microfluidic chip for selective exosome isolation. The microfluidic chip consists of Hoof Structure (HS) for mixing exosomes and two different sized aptamer-coated particles and Multi-Orifice Flow Fractionation (MOFF) for separating each particle. Methods: Biotinylated EpCAM aptamer was immobilized on the surface of 7 μm streptavidin-coated polystyrene particle and HER2 on 15 μm. The HS has the circular expansion channel on the 1st layer to generate expansion vortices and the two curvature channels on the 2nd layer to make chaotic advection. It makes transverse flow and mixes two particles without particle focusing phenomenon. The 100-nm (exosome), 7- and 15-μm fluorescence particles were used to test mixing performance between exosomes and particles in the HS. The MOFF was designed by a series of contraction/expansion microchannels for continuous size-based separation. Separation performance was tested by using the 7- and 15-μm fluorescence microparticles in the MOFF. Results: The mixing efficiency was the highest at the flow rate 150 μL/min. Each exosome was continuously captured by aptamer-conjugated particle in the HS channel. The capture efficiency of EpCAM positive exosome was 96.9% and HER 2 was 68.09%. Two particles were separated in the integrated microfluidic device at the same flow rate. Also, 96.26% of 15-μm microparticles were positioned into the centre of the channel and 89.48% of 7 μm microparticles were separated on both sides of the channel. Summary/Conclusion: Each exosome was continuously captured by mixing aptamer-conjugated particle in the HS. Exosome-conjugated microparticles were successfully separated by inertial force in MOFF. This analysis of each exosome will shed light on diagnosis and therapy of cancers. OWP3.05= PF10.11 Aqueous two-phase system to isolate extracellular vesicles for prostate cancer diagnosis Hyunwoo Shin ^a, Jiyoon Kim^a, Mee Young Kim^b, Yong Hyun Park^b, Yong Goo Kim^c, Ji Youl Lee^b and Jaesung Park^d ^aPOSTECH, Pohang, Republic of Korea; ^bDepartment of Urology, Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, Republic of Korea; ^cDepartment of Laboratory Medicine, Mary’s Hospital, The Catholic University of Korea, Seoul, Republic of Korea, Seoul; ^dDepartment of Mechanical Engineering, POSTECH, Pohang, Republic of Korea Introduction: Analysing extracellular vesicles (EVs) is an attractive means in prostate cancer diagnosis. However, existing methods of EVs isolation have low efficiency, purity and long process time, which induce low diagnostic ability. To approach the problems, we adapt a two-phase system to diagnose prostate cancer by isolating EVs from patients’ urine. Using the two-phase system, prostate hyperplasia (BPH) patients and prostate cancer (PCA) patients were diagnosed, and the diagnostic ability was compared with conventional diagnostic methods. Methods: Forty-two prostate cancer (PCA) patients and 20 benign prostate hyperplasia (BPH) patients’ urine, plasma, saliva was collected and used for identifying EVs isolation ability of aqueous two-phase system (ATPS) and for comparing diagnostic ability of ATPS with conventional diagnosis. Results: With an optimized ATPS, EVs were isolated with an efficiency of approximately 90%. In addition, the EV-isolation time was within approximately 30 min, and the purity of EVs in ATPS was approximately two times better than achieved with a conventional methods, ultracentrifugation and polymeric precipitation. After the ATPS isolated EVs from patients’ body fluid, PCR and ELISA were utilized to detect EVs derived from prostate cancer cells. The expression levels of RNA and protein markers of prostate cancer were compared, and the relationship between expression levels and clinical data was analysed. The results demonstrated that diagnostic ability based on ATPS was better than other conventional methods (serum PSA and sediments). Moreover, sensitivity increased by at least 10%, and specificity was improved by at least 20% compared to conventional methods. Summary/Conclusion: High quality and quantity of EVs can be obtained from patients’ body fluid using ATPS. Using the abundant sources, which contains cancer-related protein and genes, we can perform a diagnosis with high specificity and sensitivity. Therefore, ATPS offers a powerful tool for more specific and sensitive diagnosis. OWP3.06=PS05.11 In vitro and in vivo investigation of extracellular vesicles (EVs) as biomarker carriers in the diagnosis of early Alzheimer’s disease Soraya Moradi-Bachiller ^a, Miriam Ciani^b, Roberta Zanardini^b, Luisa Benussi^b, Roberta Ghidoni^b, J. Mark Cooper^c, Gianluigi Forloni^a and Diego Albani^a ^aDepartment of Neurosciences, Mario Negri Institute for Pharmacological Research IRCCS, Milan, Italy; ^bMolecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy; ^cDepartment of Clinical Neurosciences, Faculty of Brain Sciences, University College London – Institute of Neurology, London, UK Introduction: Extracellular vesicles (EVs) represent an ideal source of biomarkers due to their role in cellular communication and their ability to carry protein aggregates. The most investigated EVs are exosomes, active entities secreted from cells and able to cross the blood brain barrier. Several neurodegeneration-involved molecules may undergo intercellular spreading through exosome release. In Alzheimer’s disease (AD), before clinical signs appear, several proteins implicated in exo- and endocytic pathways are altered. In this scenario, the identification of a correlation between variations in proteins carried by EVs and the progression of AD is the main aim of our project. Methods: We performed exosome isolation and characterization from H4-SW glioma cells (a cell model featuring mutated β-amyloid overexpression), as well as in mouse- (triple-transgenic mouse model for familial AD) and human-plasma samples (Mild Cognitive Impairment (MCI) and AD subjects). In every case, a differential centrifugation protocol was applied and exosomes were then characterized using Nanoparticle Tracking Analysis with the NanoSight. We then explored exosome content, specifically Amyloid Precursor Protein (APP) and its proteolytic fragments, Microtubule Associated Protein Tau (tau), Progranulin (PGRN protein), Soluble Triggering Receptor Expressed on Myeloid Cells 2 (sTREM2) and α-synuclein (α-syn), using Western blot and ELISA. L1CAM and CD63 were evaluated to define the neural-derived exosomes amount in human samples. All the samples were collected after ethical committee approval respecting Helsinki’s declaration. Informed consents were provided by all the subjects. Results: Our preliminary results show that APP, PGRN and sTREM2 are carried by H4- and human plasma-derived EVs. H4-SW cell-culture medium and 3Tg mouse plasma had a decrease in the EVs number release (≈1*10e8 EVs/mL) in comparison to control (≈7*10e8 EVs/mL). This decrease was not found in human plasma samples. Summary/Conclusion: EVs purified from H4-glioma cellular AD model, 3xTg mouse-, MCI- and AD-plasma samples carry proteins relevant for neurodegenerative diseases (NDs). EVs release is reduced in cellular and animal AD-models. Funding: Horizon 2020 Marie Sklodowska-Curie Innovative Training Networks – Blood Biomarker-based Diagnostic Tools for Early Stage Alzheimer’s Disease. OWP3.07=PF12.07 Shed microvesicles released from human primary and metastatic colorectal cancer cell lines contain key cancer progression proteins and RNA species Wittaya Suwakulsiri ^a, Alin Rai^a, Rong Xu^a, Maoshan Chen^a, David Greening^b and Richard Simpson^a ^aDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia, Melbourne, Australia; ^bDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia Introduction: Extracellular vesicles (EVs) function in bidirectional cell–cell communication and contribute to the sustained growth, invasion and metastasis of cancer cells within the tumour microenvironment (TME). EVs comprise two main classes – exosomes and shed microvesicles (sMVs, also termed microparticles and ectosomes) – with distinct modes of biogenesis. Within each EV class, subtypes exist that can be distinguished by their distinct protein/ RNA signatures. Whilst much is known about exosome cargo content and functionality, sMVs are poorly understood. Methods: Here, we compare protein/ RNA profiles and functionality of sMVs and exosomes secreted from human primary (SW480) and metastatic (SW620) colorectal cancer cell lines. Milligram amounts of EVs were purified from cell culture media using a combination of differential ultracentrifugation/ isopycnic iodixanol density centrifugation. Label-free quantitative mass spectrometry was performed to obtain protein profiles for SW480-derived and SW620-derived sMVs. Results: We show that sMVs, unlike exosomes, are ALIX-, TSG101-, CD63- and CD9- and contain a different suite of key cancer progression modulators. Protein/ RNA signatures for SW480-derived sMVs and exosomes differ from each other and also from their SW620-derived counterparts. SW480-derived sMVs are enriched in ITGA/B, ANXA1, CLDN7, CD44 and EGFR/NOTCH signalling networks, while SW620-derived sMVs are enriched in PRKCA, MACC1, FGFR4 and MTOR/MARCKS signalling networks. Fibroblast invasion capabilities of SW480-derived and SW620-derived sMVs are comparable. Summary/Conclusion: Furthermore, we report for the first time a comprehensive biochemical/ functional analysis of a hitherto undescribed subpopulation of sMVs. We anticipate our in vitro findings will be a starting point for more sophisticated studies aimed at elucidating the biochemical and functional properties of EV subtypes in vivo. The emerging roles of specific EV subtypes in the TME we believe will alter our view of cancer biology and might present new targets for therapeutic intervention. Funding: Funding support from La Trobe University, Melbourne, Australia. OWP3.08=PF12.08 Mass spectrometry analysis of small extracellular vesicles isolated from ovarian cancer ascites Anna Kotrbová ^a, Kristina Gomoryova^a, David Potesil^b, Vit Weinberger^c, Igor Crha^c, Eva Jandakova^d, Lubos Minar^c, Zbynek Zdrahal^b, Vítězslav Bryja^a and Vendula Pospíchalova^a ^aDepartment of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic; ^bCore Facility Proteomics, Central European Institute of Technology, Masaryk University, Brno, Czech Republic; ^cDepartment of Obstetrics and Gynecology, Faculty Hospital Brno, Brno, Czech Republic; ^dDepartment of Pathology, University Hospital Brno, Brno, Czech Republic Introduction: High-grade serous carcinoma of the ovaries, fallopian tube and peritoneum (HGSC) is the deadliest gynaecological malignancy with 5-year survival rate below 30%. HGSC is frequently accompanied by ascites, a pathological accumulation of fluid in the peritoneum, which can be exploited as a liquid biopsy containing not only cancer cells, but also the tumour microenvironment including extracellular vesicles (EVs). Tumour cells produce substantially more EVs than healthy cells, thus malignant ascites is the source of enriched pool of EVs of HGSC origin. Methods: Ascitic fluids depleted of cells were fractioned using size-exclusion chromatography and two fractions – containing and not containing EVs – were further analysed. In parallel, small EVs were also isolated from ascitic fluids using differential ultracentrifugation followed by purification step in sucrose/D2O cushion. In total, 24 malignant ascites and 5 non-malignant ascites were used for EV isolation and further analysed using high-resolution hybrid mass spectrometer Orbitrap Fusion Lumos Tribrid. The subsequent data visualization and statistical analyses were performed using in-house-developed pipelines in KNIME environment. Results: We identified 2441 proteins, in total, in the EVs from the ascites among which 21 were present in all 29 EV samples and not in non-vesicular fractions. Several of these proteins were specifically enriched in small EVs in malignant ascites in comparison with non-malignant ascites. These proteins are now being evaluated as biomarkers. Summary/Conclusion: Using advanced mass spectrometry, we identified candidate proteins which are specifically enriched in small EVs of HGSC. These proteins warrant further investigation as they may act as important players in HGSC progression as well as serve as potential prognostic/diagnostic/screening biomarkers of HGSC. Funding: Czech Science Foundation, Grant No. GJ17-11776Y. OWP3.09=PT09.12 Identification of single tumour-derived extracellular vesicles by means of optical tweezers and Raman spectroscopy Agustin Enciso-Martinez ^a, Edwin van der Pol^b, Aufried Lenferink^c, Leon Terstappen^a and Cees Otto^a ^aMedical Cell Biophysics, University of Twente, Enschede, Netherlands; ^bAmsterdam UMC, University of Amsterdam, Department of Biomedical Engineering and Physics, Amsterdam, Netherlands, Amsterdam, Netherlands; ^cUniversity of Twente, Enschede, Netherlands Introduction: EVs derived from cancer cells play a role in tumour cell proliferation, migration, invasion and metastasis. Their presence in body fluids, such as blood, makes them potential biomarkers for cancer disease. However, the identification of single tdEVs can be challenging due to their heterogeneity, their ultra-small size, their size overlap with many other normal EVs and contaminants in body fluids and the lack of knowledge on their chemical composition. Methods: Synchronized optical tweezers and Raman spectroscopy have enabled a study of individual EVs. The new method detects individual trapping events from Rayleigh scattering. The synchronous recording of Raman scattering enabled the acquisition of Raman spectra of both individual and multiple EVs, disclosing their chemical composition. Furthermore, Mie light scattering theory has been used to relate the Rayleigh scattering intensity to the size of trapped EVs. Results: The light scattered of trapped EVs gave rise to step-wise time traces that can be used to distinguish individual trapping events from accumulative cluster events due to the discrete nature of the steps which correspond to single trapping events. Next, we confirmed the trapping of individual EVs derived from PC3 cells, red blood cells, platelets and blood plasma by acquiring both, Rayleigh and Raman scattering signals. While the step-wise trend in the Rayleigh scattering signal suggests trapping of single particles, the Raman scattering signal demonstrates the nature of the trapped EVs. Through principal component analysis (PCA), the main spectral variations among the four EV types were identified. The principal component scores grouped the PC3-derived EVs in a separate cluster from the rest of the EVs. Summary/conclusion: We have developed an automated single particle optical tweezers – Raman and Rayleigh scattering setup to trap and release single EVs over time. We demonstrated single-EV trapping by simultaneous acquisition of Rayleigh and Raman scattering. PCA enabled the identification of single-EVs derived from the cancer cell line PC3. This discloses chemical information as a step towards the identification and characterization of single tumour-derived EVs in blood. Funding: Cancer ID – project number 14193, (partially) financed by the Netherlands Organisation for Scientific Research (NWO) Symposium Session 5: EVs in Infectious Diseases Chairs: Shilpa Buch; Vera TangLocation: Level B1, Hall A 14:15–15:00 OT05.01 Extracellular vesicles provide a capsid-free vector for oncolytic adenoviral DNA delivery Heikki Saari ^a, Tiia Turunen^b, Mikko Turunen^b, Matti Jalasvuori^c, Sarah Butcher^d, Seppo Ylä-Herttuala^b, Tapani Viitala^a, Vincenzo Cerullo^a, Pia Siljander^e and Marjo Yliperttula^a ^aDivision of Pharmaceutical Biosciences and Drug Research Program, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland; ^bA.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland; ^cDepartment of Biological and Environmental Science, Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland; ^dFaculty of Biological and Environmental Sciences, Molecular and Integrative Bioscience Research Programme, and Helsinki Institute of Life Sciences, Institute of Biotechnology, University of Helsinki, Helsinki, Finland; ^eEV-group, Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland Introduction: Extracellular vesicles (EVs) have been showcased as auspicious candidates for delivering therapeutic cargo, including oncolytic viruses for cancer treatment. Delivery of oncolytic viruses in EVs could provide considerable advantages, hiding the viruses from the immune system and providing alternative entry pathways into cancer cells. Here we describe the secretion and viral cargo of EVs secreted by cancer cells infected with an oncolytic adenovirus (IEVs, infected cell-derived EVs) as a function of time after infection. Methods: IEV-containing cell culture medium was collected from A549 and PC-3 cancer cell cultures every 24 h after being infected with an oncolytic adenovirus and IEVs were isolated by iodixanol density gradient centrifugation. IEVs were then characterized by cryo-TEM, NTA, immunoblotting and qPCR for structural properties and viral components and their infectivity was confirmed by cytotoxicity assay and TEM of IEV-treated cells. Results: IEVs were secreted already before the lytic release of virions and their structure resembled normally secreted EVs, suggesting that they were not just apoptotic fragments of infected cells. IEVs were able to carry the viral genome and induce infection in other cancer cells. The amount of viral cargo associated with IEVs increased as the infection progressed, although no intact virions were observed in any of the IEVs visualized by cryo-TEM. The amount of viral cargo also appeared to be density-dependent, in that heavier IEVs contained more viral DNA and protein per vesicle. Summary/Conclusion: Given that adenovirus is a DNA-virus that is assembled in the nucleus and released via induced lytic cell death, the generation of infective EVs as prescribed here suggests that part of the produced viral DNA is secreted via IEVs before cell lysis. As such, the role of EVs in the life cycle of adenoviruses may be an important part of the successful infection and may also be harnessed for cancer- and gene-therapy as vectors of viral DNA invisible to the immune system. OT05.02 Bacterial growth stage regulates the size, composition and biological functions of membrane vesicles Lauren Zavan^a, Natalie Bitto^a, Ella Johnston^a, David Greening^b and Maria Kaparakis-Liaskos ^a ^aDepartment of Physiology, Anatomy and Microbiology, La Trobe University, Melbourne, Australia; ^bDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia Introduction: Outer membrane vesicles (OMVs) are naturally released by all Gram-negative bacteria as part of their normal growth and contain many of the components found in their parent bacterium, including DNA, RNA and proteins. To date, few studies have compared the proteome of OMVs to that of their parent bacterium and examined how it changes throughout bacterial growth. In this study, we aimed to elucidate the contribution of bacterial growth stage on the size, composition and biological functions of Helicobacter pylori OMVs. Methods: OMVs were purified from H. pylori cultures grown to early log, mid log or stationary phase of bacterial growth, and their size and protein composition were analysed using NTA and proteomics, respectively. The ability of OMVs isolated from various growth stages to stimulate an inflammatory response in human epithelial cells was determined by ELISA. Results: We found that OMVs became less heterogeneous in size throughout bacterial growth. We showed that the proteome of OMVs was vastly different to that of their parent bacterium from each time point, suggesting that there is preferential cargo packaging of bacterial proteins into OMVs. Gene ontology and enrichment analyses identified that bacterial growth stage regulated the type of proteins packaged into OMVs, as early log and stationary phase OMVs were enriched in proteins required for metabolic pathways, whereas late log phase OMVs contained proteins contributing to cell signalling. Finally, we identified that bacterial growth stage affected the inflammatory response mediated by OMVs in host epithelial cells, highlighting that bacterial growth stage regulates the subsequent biological functions of OMVs. Summary/Conclusion: Our findings identify that bacterial growth stage regulates the size, protein cargo composition and biological functions of H. pylori OMVs, and that therefore OMVs from various growth stages are not comparable. Collectively, these findings emphasise the importance of considering bacterial growth stage from which OMVs are isolated from, as this will ultimately affect their protein content and biological functions. We are currently determining whether bacterial growth stage also regulates the composition and functions of Gram-positive bacterial membrane vesicles. Funding: Australian Research Council. OT05.03 Can exosomes be used to predict where patients are on the tuberculosis disease spectrum? Nicole Kruh-Garcia, Gustavo Diaz, Cristian Oliva Aviles and Karen Dobos Colorado State University, Fort Collins, USA Introduction: Mycobacterium tuberculosis (Mtb), the causative agent of the disease tuberculosis (TB), is a highly successful human pathogen. Mtb has the ability to survive within the host macrophage. In response to the challenges of the intracellular environment, the bacteria secretes a dynamic subset of proteins reflective of its metabolic state, some of which are entrapped in exosomes and released from the host cell. Our ultimate goal is to improve upon the current diagnostics to facilitate early and rapid diagnosis of active disease, which is a key to timely drug invention and further spread of the disease. Using serum exosomes, we aimed to determine if a proteomic fingerprint can be used to discriminate between individuals with TB from non-TB, as well as to classify TB suspects – individuals with pulmonary symptoms but without detectible mycobacteria in their sputum. Methods: Hyper Reaction Monitoring Mass Spectrometry (HRM-MS) was applied to a sample set of serum exosomes isolated from four groups: TB negative (healthy non-endemic and TB-suspect endemic) and TB positive (smear negative or positive, all culture positive) individuals. This allowed us to decipher a host protein profile that is common with exosomes from individuals who have sputum confirmed TB and distinct from those of whom do not. Peptide intensities were normalized and differences were in abundance, which were determined by t-test. Results: Nine proteins – including FCGR3A and α-2-HS-glycoprotein, show distinct patterns, either increasing or decreasing with disease severity. Using adaptive least absolute shrinkage and selection operator (Lasso) we are able to discriminate TB patients from healthy and TB suspects using nine proteins. Application of the model to a test set of smear and culture negative TB suspect serum exosomes, we found that nine were consistent with the negative diagnosis, while one surpassed the threshold for positivity. When the same sample was screened for the presence of mycobacterial peptides using our published targeted MS assays, it was positive for several Mtb peptides. Summary/Conclusion: These results indicate that a novel assay detecting a combination of host and Mtb proteins can discriminate TB positivity with greater sensitivity than the current sputum diagnostics. Funding: Bill and Melinda Gates Foundation Fund #: OPP1039688. Symposium Session 6: EV Engineering I Chairs: Hang Hubert Yin; Siyang ZhengLocation: Level 3, Hall B 13:30–15:00 OT06.01 Designed EVs for intracellular delivery of therapeutic antibodies Oscar Wiklander ^a, Dhanu Gupta^a, Joel Nordin^a, Heena Sharma^b, Xiuming Liang^a, Giulia Corso^a, Dara Mohammad^a, Rim Jawad^a, André Görgens^c and Samir El Andaloussi^d ^aClinical Research Center, Department for Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; ^bEvox Therapeutics Limited, Oxford, UK; ^cKarolinska Institutet, Department of Laboratory Medicine, Stockholm, Sweden; ^dDepartment of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden Introduction: Extracellular vesicles (EV) can be engineered to display various targeting and therapeutic moieties. Their ability to also act as a natural vector to shuttle cargo over biological barriers offers a unique platform for the development of a new class of therapeutics. Here, we introduce a novel concept consisting of antibody coupled therapeutic EV in order to target tissues or intracellular pathways. Methods: By engineering EV to express an Fc-binding moiety (Fc-EV), antibodies can be displayed on the surface of the vesicles. We have extensively evaluated the capacity of these EV to bind antibodies by immuno-electron microscopy, cellular uptake of labelled antibodies/EV and flow cytometry analysis, which indicates that EVs can be decorated with antibodies. As a proof of concept, antibodies bound to Fc-EV, were assessed in inflammatory models as well as in cancer settings. Results: Delivery of anti-STAT3 antibodies in an in vitro STAT3 dependent inflammatory reporter model was assessed, with promising results showing inhibition of STAT3 transcriptional activity. Furthermore, intracellular delivery of anti-STAT3 antibody using Fc-EV displays a dose dependent growth inhibition in pancreatic ductal adenocarcinoma (PDAC) cells. The Fc-EV platform can also be utilized for decorating EVs with cancer targeting antibodies, a feature that can be harnessed to address the differences in uptake displayed by different cancers. Certain cancer types are known to rapidly internalize EV, whereas other cancer types, such as malignant melanoma are known to take up EV to a very low extent, if taken up at all. Our results show that antibodies targeting surface molecules of cancer cells also aid the internalization of EV into cancer cells, thus further indicating the potential of utilizing EV as therapeutic vectors. In order to achieve specific targeting to B16F10 malignant melanoma cells, we have decorated the EV surface with antibody targeting surface proteins that are known to be displayed on B16F10 cells, which lead to cellular association of EV to these cells. Summary/Conclusion: Overall the Fc-EV platform offers the prospective of combining antibody and EV technology, with potential applications including tissue and cell targeting as well as intracellular delivery of functional antibodies. OT06.02 Extracellular vesicles derived from AT-MSCs mediated miR-424 delivery promote apoptosis via the PD-L1/PD-1 pathway in TNBC Yueyuan Zhou ^a, Nobuyoshi Kosaka^b, Zhongdang Xiao^c and Takahiro Ochiya^b ^ayueyuanzhou16@hotmail.com, Tokyo, Japan; ^bDepartment of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Shinjyuku-ku, Japan; ^cSoutheast University, Nanjing, China (People’s Republic) Introduction: Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) showed great potential as the delivery vehicle of drugs including miRNAs based on its low immunogenicity and natural homing ability. Triple-negative breast cancer (TNBC) is an aggressive and invasive subtype that has limited treatment options. Meanwhile, TNBC is immunogenic with a greater percentage of tumour-infiltrating lymphocytes and increased expression of the programmed death-ligand 1 (PD-L1) in the tumour microenvironment. The aim of our study is to apply MSC-EVs to modulate the expression of PD-L1 via the delivery of miR-424 and contribute to the immunotherapy for TNBC. Methods: EVs generated from adipose tissue-derived MSCs (AT-MSCs) were isolated by differential centrifugation and characterized by western blot, nanoparticle tracking analysis and transmission electron microscopy. Before EV collection, AT-MSCs were modified to overexpress miR-424 through electroporation, and miRNA mimics transfection. The miRNAs targeting PD-L1 was predicted according to in silico analysis. The direct regulation of miR-424 on PD-L1 was verified via the 3’-UTR luciferase report assays. The purified EVs were added to the recipient MDA-MB-231 cells (MM-231). The expression of PD-L1 mRNA and protein was analysed via qRT-PCR and western blot, respectively. Results: We found that miR-424 directly regulated the expression of PD-L1 through the binding to PD-L1 3’UTR. Furthermore, the expression of PD-L1 in MM-231 cells was down-regulated and the expression of miR-424 in MM-231 was up-regulated after coculture with exosomes derived from normal AT-MSCs, and AT-MSCs with miR-424 overexpression. Moreover, the cell viabilities of MM-231 were decreased after coculture with exosomes or transfected with miR-424 mimics. Summary/Conclusion: EVs derived from AT-MSCs could transfer functional miR-424 to TNBC cell lines and promote the apoptosis via decreased immune-negative PD-L1/PD-1 pathway. Funding: This work was supported by Project for Cancer Research and Therapeutic Evolution [P-CREATE; grant number:17cm0106402h0002], MEXT KAKENHI [Grant-in-Aid for Young Scientists (A); grant number: 17H04991] and China Scholarship Council [grant number: 201706090122]. OT06.03 Exosomal delivery of NF-κB repressor delays LPS-induced preterm birth in mouse models Samantha Sheller-Miller ^a, Kyungsun Choi, George Saade, Chulhee Choi^b and Ramkumar Menon ^aUniversity of Texas Medical Branch, Galveston, USA; ^bKAIST, Daejeon, Republic of Korea Introduction: Intraamniotic infection and inflammation are associated with spontaneous preterm birth (PTB) and preterm premature rupture of the membranes (pPROM). In this study, we tested engineered extracellular vesicles, or exosomes, cargoing an inhibitor to pro-inflammatory transcription factor (NF-kB), called super-repressor (SR) IkB, to prolong gestation in an infection (LPS)-induced PTB mouse model. Methods: HEK293T (human embryonic kidney cell) derived exosomes were engineered to contain SR using a protein loading via optically reversible protein–protein interaction (EXPLORs) method (Yim, et al 2016). In this method, SR is actively incorporated into exosomes during biogenesis. These exosomes were isolated, quantified and used for our studies. Intraperitoneal (IP) injection of either LPS (100 g) or PBS were performed in CD-1 mice on gestational day 15 followed by injection of PBS, SR exosomes (1 × 1010) or naïve exosomes (exosomes derived from HEK293T cells under normal culture conditions, 1 × 1010) every 2 h for a total of five injections. Treatment groups (Group 1-LPS+PBS; Group 2-LPS+SR; Group 3-LPS+naïve, and Group 4-PBS) were monitored for preterm birth. Upon delivery of at least one pup in Group 1, mice were euthanized, and maternal plasma, uterus and cervix were collected for cytokine analysis using Luminex (IL-1β, IL-8 and IL-10) and Western blot for NF-B activation via RelA phosphorylation (P-NF-B), respectively. Survival graphs were created in GraphPad and one-way ANOVA was performed to determine statistical significance (P < 0.05). Results: Animals injected with PBS delivered at the expected gestational age (19.5 days). LPS and LPS + naïve-induced PTB within 10 h; however, injection of SR exosomes prolonged delivery by an average of 21 h in this model. Consistently lower levels of pro-inflammatory cytokines, IL-1β and IL-8, were seen in maternal plasma of LPS + SR compared to LPS mice, while anti-inflammatory cytokine, IL-10, levels were significantly increased in LPS + SR mice compared to LPS (P = 0.01) and PBS controls (P < 0.0001). In the cervix and uterus, P-NF-B expression was significantly decreased in LPS + SR compared to LPS (P = 0.005, P = 0.03) (Figure 2B). Summary/Conclusion: Exosomes can be engineered to carry pharmaceutical agents that can dampen the infection-induced inflammation associated with PTB and pPROM. OT06.04 Technologies for loading RNA-based therapeutics into extracellular vesicles for drug delivery Olga Shatnyeva ^a, Anders Gunnarsson^b, Euan Gordon^c, Elisa Lázaro-Ibáñez^d, Lavaniya Kunalingam^c, Xabier Osteikoetxea^e, Kristina Friis^c, Marcello Maresca^c and Niek Dekker^b ^aAstraZeneca, Molndal, Sweden; ^bAstraZeneca, Mölndal, Sweden; ^cAstrazeneca, Mölndal, Sweden; ^dAstraZeneca, molndal, Sweden; ^eAstraZeneca, Macclesfield, UK Introduction: Extracellular vesicles (EVs) have emerged as a very potent new delivery system for drug delivery. Recent advances in RNA-based therapeutics have broadened the scope of cellular targeting of currently undruggable genes. Current approaches for RNA loading of EVs suffer from poor efficacy. Our study combines bioengineering of the therapeutic EVs with post-isolation RNA. We will here present data showing (1) the use of RNA binding proteins (RBP) fused to EV protein markers for in vitro loading of EVs with tagged RNA cargo and (2) post-isolation incubation of EVs with RNA-loaded lipid nanoparticles (LNP). Methods: A library of targeted RNAs fused to a specific RNA binding protein (RBP) sequence was generated, varying the position of recognition site. Surface plasmon resonance was used to characterize the modified sgRNAs for binding to the RBP. Activity of the hybrid sgRNA was also confirmed for functional gene editing with Cas9. Expi293F cells were co-transfected with the set of modified sgRNAs and RBP fused to EV proteins followed by EV purification by differential ultracentrifugation. EVs were characterized by nanoparticle tracking analysis, Western blotting and single molecule microscopy. Efficiency of sgRNA loading into EVs was determined using qPCR. Post-isolation loading of sgRNA with Expi293 EVs by co-incubation and functional delivery of sgRNA cargo in HEK293 cells were also evaluated. Results: The introduction of RNA recognition elements into sgRNA sequence did not interfere with binding to RBP. Fusions between RBP and EV proteins resulted into efficient incorporation of RBP in EVs. Co-expression of sgRNA resulted in selective targeting of sgRNA to EVs. Additionally, EVs from cells co-expressing sgRNA and RBP contained 10-fold more sgRNA compared to EV from cells who only expressed sgRNA. Loading of synthetic sgRNA cargo with 40% encapsulation efficiency was achieved by incubation of EVs with LNPs and the resulting particles led to functional uptake in HepG2 cells. Summary/Conclusion: Here, we compare different techniques for therapeutic cargo loading and delivery into target cells. All approaches for RNA loading into EVs demonstrates proof of principle. We envision that this approach will be useful for RNA loading for therapeutic applications. OT06.05 Engineering designer exosomes produced efficiently by mammalian cells in situ and their application for the therapy of Parkinson’s disease Ryosuke Kojima ^a, Daniel Bojar^b and Martin Fussenegger^c ^aGraduate School of Medicine, The University of Tokyo. JST PRESTO, Tokyo, Japan; ^bETH Zurich, Department of Biosystems Science and Engineering, Basel, Switzerland; ^cETH Zurich, Department of Biosystems Science and Engineering. University of Basel, Faculty of Science, Basel, Switzerland Introduction: Exosomes are cell-derived extracellular nanovesicles 50–150 nm in size, which serve as intercellular information transmitters in various biological contexts, and are candidate therapeutic agents as a new class of drug delivery vesicles. However, inefficiency of exosome cargo transfer, such as transfer of mRNA contained in exosomes, and lack of methods to create designer exosomes has hampered the development of sophisticated therapeutic interventions. Methods: We have developed a set of synthetic-biology-inspired genetic devices that enable efficient customizable in situ-production of designer exosomes in engineered mammalian cells, and pursued their therapeutic applications. Results: The developed synthetic devices that can be genetically encoded in exosome producer cells (named “EXOtic (EXOsomal Transfer Into Cells) devices”) enhance exosome production, specific mRNA packaging and delivery of the mRNA into the cytosol of recipient cells. Synergistic use of these devices with a targeting moiety significantly enhanced functional mRNA delivery into recipient cells, enabling efficient cell-to-cell communication without the need to concentrate exosomes. Further, the engineered exosome producer cells implanted in living mice could consistently deliver mRNA to the brain. Moreover, therapeutic catalase mRNA delivery by designer exosomes attenuated neurotoxicity and neuroinflammation in both an in vitro and in vivo Parkinson’s disease model. Summary/Conclusion: These results indicate the potential usefulness of the EXOtic devices for RNA delivery-based therapeutic applications. (Nat. Commun. 2018, 9, 1305) Funding: This work was supported by the European Research Council (ERC) advanced grant [ProNet, no. 321381] and in part by the National Centre of Competence in Research (NCCR) for Molecular Systems Engineering (to M.F.). R.K. was supported by a postdoctoral fellowship from the Human Frontier Science Program. OT06.06 Protein engineering for loading of Extracellular Vesicles Xabier Osteikoetxea ^a, Josia Stein^a, Elisa Lázaro-Ibáñez^b, Gwen O´Driscoll^c, Olga Shatnyeva^d, Rick Davies^a and Niek Dekker^c ^aAstraZeneca, Macclesfield, UK; ^bAstraZeneca, molndal, Sweden; ^cAstraZeneca, Mölndal, Sweden; ^dAstraZeneca, Molndal, Sweden Introduction: To date various reports have shown the utility of extracellular vesicles (EVs) for delivery of therapeutic protein cargo. Currently, the most common strategies for loading therapeutic cargoes occur after EV isolation mixing EVs with desired cargo and subjecting to passive incubation, electroporation, freeze-thaw cycling, sonication, extrusion, or membrane permeabilization with saponin among various techniques. An alternative approach is to modify releasing cells to secrete EVs containing the desired cargo with minimal impact on native EVs by post-isolation treatments. In this study, we designed different constructs to compare Cre and Cas9 loading efficiency into EVs using (1) light-induced dimerization systems (Cryptochrome 2 (CRY2), Phytochrome B (PHYB), and Vivid-based “Magnets” (Mag)), (2) fusion to EV associated proteins (CD9, CD63, CD81, Rab5), (3) lipidation motifs (Myristoylation, Palmitoylation, Prenylation) and (4) a novel motif “mXO”. Methods: For EV production human Expi293F cell line was transiently transfected with plasmid constructs using PEI MAX 40K. EVs were then isolated by differential ultracentrifugation followed by iodixanol gradient and characterized using nanoparticle tracking analysis, western blotting and transmission electron microscopy. Functionality of engineered Cre and Cas9 cargo was assessed in reporter cell assays using fluorescent microscopy, qPCR and Sanger sequencing followed by TIDE analysis. Results: Light-induced dimerization using CRY2 resulted in better EV cargo loading for both Cre and Cas9 than PHYB and Mag systems. Among the EV associated proteins and lipidation motifs tested CD9, CD81, Myristoylation and mXO were most efficient at recruiting Cre and Cas9 cargo by light-induced dimerization. Using CRY2 in combination with CD9 or mXO we achieved loading efficiencies of 10–34 Cre molecules per EV and 23–30 Cas9 molecules per EV. Summary/Conclusion: Cre and Cas9 loading into EVs by producing cells is feasible using protein engineering with light-induced dimerization. Of the designs investigated CRY2-induced light dimerization and CD9 and mXO motif were most effective resulting in multiple copies of functional Cre and Cas9 loaded per EV. We envision that this approach will be useful for protein loading in a variety of therapeutic applications. OT06.07 Engineered extracellular vesicles for drug delivery Niek Dekker^a, Elisa Lázaro-Ibáñez^b, Olga Shatnyeva^c, Nikki Heath^d and Xabier Osteikoetxea^e ^aAstraZeneca, Mölndal, Sweden; ^bAstraZeneca, molndal, Sweden; ^cAstraZeneca, Molndal, Sweden; ^dAstraZeneca, Vancouver, Canada; ^eAstraZeneca, Manchester, United Kingdom Abstract: Extracellular Vesicles (EVs) represent an exciting opportunity as biological delivery vehicle for therapeutic cargo with excellent safety, low intrinsic immunogenicity, cell-specific tropism and biological delivery efficiency. Methods: There are multiple approaches for the introduction of protein and RNA cargo into EVs, including physical, chemical and cell engineering. We have engineered Expi293F suspension cells with transient expression of fusion proteins for reversible loading with protein cargo with examples for Cre recombinase and Cas9 for CRISPR gene editing. Results: We have developed a single molecule fluorescence microscopy technique to quantify cargo loading at a single particle level, showing excellent loading for GFP fusions with CD63 with on average 70 copies of the fusion protein per particle. Functional delivery of Cre recombinase, as measured in a reporter cell line, was dependent on addition of small molecule or peptide enhancers of endosomal escape. Using RNA-binding proteins fused to exosomal markers we were able to enrich EVs with RNA cargo with up to 10-fold higher loading of sgRNA compared to loading from passive mass redistribution. Human Expi293F cell-derived EVs did not trigger any significant immune response in vitro in human blood. The in vivo assessment following a single intravenous administration of these EVs in BALB/c mice did not reveal marked haematological changes, cytokine induction or histopathological effects. Labelling of EVs using fluorescent mCherry, luminescent NanoLuc or radio-isotope 111Indium marker allowed for on line analysis of bio-distribution in vivo. Summary/conclusion: Opportunities of naïve and engineered EVs for drug discovery and their potential for therapeutic applications will be discussed. PT01: Cellular and Organ Targeting Thursday Poster SessionChairs: Charles Lai; Ikuhiko NakaseLocation: Level 3, Hall A15:30–16:30 PT01.01 Role of circulating extracellular vesicles in brain function and behaviour Eisuke Dohi, Indigo Rose, Takashi Imai, Rei Mitani, Eric Choi, Dillon Muth, Zhaohao Liao, Kenneth Witwer and Shinichi Kano Johns Hopkins University School of Medicine, Baltimore, USA Introduction: Accumulating evidence suggests that extracellular vesicles (EVs) circulate in the blood and affect cellular functions in an organ distant from their origins. In neuroscience, systemic circulating factors such as cytokines/chemokines, hormones and metabolites have been shown to modulate brain function and behaviour. They are also utilized as biomarkers to reflect brain disease status. Nonetheless, it remains unclear whether circulating EVs modulate brain function and behaviour. Methods: We used mouse models to study the effects of EVs from specific cell types on brain function and behaviour. Because circulating EVs are extremely heterogeneous, we focused on immunodeficient mice that lack specific lymphocytes (T and B cells). We assessed the changes in their circulating EVs and examined their potential impact on the corresponding behavioural and neuronal dysregulation. Results: As expected, immunodeficient mice lack the expression of T and B cell-related markers in the EV containing fractions from the peripheral blood. Immunodeficient mice also displayed social behavioural deficits, accompanying by enhance c-Fos immunoreactivity in the excitatory neurons in the medial prefrontal cortex (mPFC). Notably, transfer of splenocytes from wild-type (WT) rescued the behavioural deficits, serum EVs and brain c-Fos expression patterns in immunodeficient mice. Further analysis on the molecular mechanisms is in progress. Summary/Conclusion: Our study has revealed a potential periphery-brain communication via EVs under physiological condition. Future studies are required to identify the cellular targets of circulating EVs and their ascending routes in the brain. Funding: NIMH R01. PT01.03 In vivo tracking and monitoring of extracellular vesicles with a new non-lipophilic dye Sam Noppen ^a, Gareth R Willis^b, Antonios Fikatas^a, Archana Gupta^c, Amirali Afshari^c, Christophe Pannecouque^a and Dominique Schols^a ^aLaboratory of Virology and Chemotherapy, Rega Institute, KU Leuven, Leuven, Belgium; ^bDepartment of Pediatrics, Harvard Medical School, MA, Boston, USA; ^cSystem Biosciences (SBI), Palo Alto, CA, USA Introduction: Extracellular vesicles (EVs) are gaining increasing interest as drug delivery vehicles. However, there is still a lack of knowledge about the in vivo fate of exogenous delivered EVs. Noninvasive optical imaging is an important tool to analyse the biodistribution of EVs. Currently, one of the most popular techniques is to directly label EVs with fluorescent lipophilic dyes. A major drawback is that the dye itself rather than EVs is detected. Hence, there is a need for other dyes that overcome these limitations. A new non-lipophilic near infrared (NIR) dye, ExoGlow-Vivo (SBI), was tested in vivo in mice. Methods: EVs from human PBMC, HEK and MCF7 cells were labelled with ExoGlow-Vivo, precipitated with Exoquick-TC (SBI) and injected intravenously (i.v.) in adult SCID mice. Human mesenchymal stem cell (MSC)-derived EVs were labelled with ExoGlow-Vivo dye, washed via ultracentrifugation and injected i.v. in post-natal day-4 FVB mice. Fluorescent images were acquired with an IVIS® Spectrum (PerkinElmer). Results: Spectral unmixing of ExoGlow-Vivo revealed optimal excitation/emission at 745/820 nm. Biodistribution studies in SCID mice showed the liver as main targeted organ. A high fluorescent signal was measured in the bladder but almost completely disappeared within 4 h. Ex vivo imaging of dissected organs confirmed the liver as the main organ, followed by spleen and kidneys. No difference in biodistribution was observed between PBMC, HEK and MCF7-derived EVs. MSC-derived EVs accumulated preferentially in the liver of post-natal mice. Interestingly, ex vivo imaging revealed high positive staining in the lungs. This may be associated with recent observations that found MSC-derived EVs ameliorate core features of experimental bronchopulmonary dysplasia. Summary/Conclusion: ExoGlow-Vivo has excellent NIR properties for in vivo imaging with almost negligible background. This non-lipophilic dye is ideal for biodistribution and kinetic studies of exogenous delivered EVs. However, uptake of most EVs by liver supplants the signal of other organs. PT01.04 Exosomal lipids applicable to cancer targeting Yuki Toda ^a, Saeka Ukai^a, Akari Kobayashi^a, Shin-ya Morita^b, Shigekuni Hosogi^a and Eishi Ashihara^a ^aDepartment of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto, Japan; ^bDepartment of Pharmacy, Shiga University of Medical Science Hospital, Kyoto, Japan Introduction: Cancer-targeting technologies must be a crucial technology to develop diagnosis and therapy of cancers. Many basic and clinical studies highlighted lots of cancer-associated proteins and expected their ligands to honour the drug carrier with selectivity; however, their targeting potential were not tolerable of advanced trials. We have previously identified glioblastoma-derived exosomes (Exo-U251) that were more effectively internalized into some types of cancer cell lines than into non-cancer cells. Because this tropism was still maintained in the lack of protein ligand interaction of the exosomes with cells, we focused on their lipid components. Methods: Ultrapure exosomes (about 100 nm vesicles expressing CD63 in d = 1.16 mg/L fraction) were collected from cell culture media using density-gradient ultracentrifugation. Exosomal lipids were extracted by Bligh & Dyer method and reconstructed into liposomes (Exolip-U251) using extrusion method. Each amount of lipid components was analysed by enzymatic fluorometric assay [Morita S.Y. and Terada T. Sci. Rep., 5:11737, 2015]. Exosomes or reconstructed liposomes were fluorescent-labelled and applied to cancer or non-cancer cells to evaluate the internalization efficiency using an image analysis of a laser scanning microscopy. Exolip-U251 conjugating siRNA was prepared by Exo-Fect reagent. Doxorubicin (DOX) was encapsulated into liposomes using remote-loading method. Results: The enzymatic fluorometric assays revealed the uniqueness of the exosomal lipid components according to the cells from which they are derived. The tropism of Exo-U251 lipid-reconstructed liposomes (Exolip-U251) partly mimicked that of the original exosomes. The siRNA conjugated Exolip-U251 was effectively delivered into the inside of cells, but did not suppress the target gene expression. By contrast, DOX-loaded Exolip-U251 significantly suppressed the proliferation of U251 cells. Thus, the encapsulation was critical for the agents to internalize more effectively into cancer cells. Summary/Conclusion: The tropism of exosomes is partially regulated by their own lipid components and, mimicking this approach would lead to promising cancer-targeting technologies. Funding: MEXT-Supported Program for the Strategic Research Foundation at Private Universities PT01.05 The exosome that are released from mechanical stress-stimulated osteocyte induces osteoclastegenesis Tomohiro Itoh ^a and Yukihiro Akao^b ^aGraduate School of Bioresources, Mie University, Tsu, Japan; ^bGifu University Graduate School of Drug Discovery and Medical Information Sciences, Gifu, Japan Introduction: Osteocyte, which is the most abundant cell in bone tissues, is well known as a mechanical stress receiving cell. During bone remodelling, bone resorptionby osteoclasts precedes bone formation by osteoblasts. However, its mechanism is still unknown. In this study, we examined whether exosome released from osteocyte by MS stimulation are involved in osteoclast differentiation. Methods: MC3T3-E1 cells or MLO-Y4 cells were seeded on 3D scaffold and grown to 70–80% confluence. The cells were exposed to pressure of 1.5 MPa for 1 h at 37°C consisting a hydrostatic pressure system. After cultivation, the cultured media harvested and then isolated then centrifuged at 8,000 ×g for 30 min at 4°C to remove cell debris. The extracellular exosomes were pelleted in a final ultracentrifugation at 100,000 ×g for 1 h at 4°C. Pelleted exosomes were resuspended in PBS and ultracentrifuged again. The size distribution of exosomes was examined using a NanoSight Tracking Analysis LM20 System. The amount of osteoclast differentiation was estimated by TRACP staining. The MLO-Y4 cell vesicle membrane and vesicle internal protein profiles were analysed by nano-LC-MS/MS based shotgun proteomics. Results: The vesicles isolated from mechanical stress-loaded MC3T3-E1 cells facilitated the mechanical stress-loaded osteoblast differentiation, but no effect against normal MC3T3-E1 cells. Though the vesicles isolated from mechanical stress-loaded MLO-Y4 cells had no effect against osteoblast differentiation, these vesicles significantly induced osteoclast differentiation. To characterize the mechanisms by which mechanical stress-loaded MLO-Y4 cell vesicles induces osteoclast differentiation in murine macrophage RAW264 cells, we analysed vesicle membrane and vesicle internal proteins by nano-LC-MS/MS-based shotgun proteomics. As a result, Protein X was only detected in mechanical stress-loaded MLO-Y4 cell vesicles. Summary/Conclusion: Our data indicated that mechanical stress-loaded MLO-Y4 cells vesicles are acting as one of osteoclast differentiation mechanisms. Now, we are further investigating whether Protein X is involved in osteoclast differentiation. Funding: This work was supported by a Grant-in-Aid for Scentific Research (C) [No. 18K11019] from Japan Society for the Promotion of Science (JSPS). PT01.06 A label-free aptasensor for electrochemical detection of gastric cancer exosomes lI Zhiyang ^a and He Nongyue^b ^aNanjing Drum Tower Hospital Clinical College, Nanjing, China (People’s Republic); ^bSoutheast University, Nanjing, USA Introduction: Emerging evidence indicates exosomes derived from gastric cancer cells enhances tumour migration and invasion through the modulation of tumour microenvironment. Here we represent a label-free electrochemical aptasensor for specific detection of gastric cancer exosomes. This platform contains an anti-CD63 antibody modified gold electrode and a gastric cancer exosome specific aptamer. The aptamer is linked to a primer sequence which is complementary to a G-quadruplex circular template. The presence of target exosomes could trigger rolling circle amplification and produce multiple G-quadruplex units. This HRP mimicking DNAzyme could catalyses the reduction of H[2]O[2] and generate electrochemical signal. This aptasensor exhibits high selectivity and sensitivity towards gastric cancer exosomes with a linear response range from 4.8 × 10^3 to 4.8 × 10^6 exosomes/mL. Therefore, we expect this electrochemical apatasensor to become a useful tool for the early diagnosis of gastric cancer. Methods: First of all, several gastric cancer cell or cancer overexpressed protein aptamers were screened in order to select gastric cancer exosome specific aptamer. Then different kinds of exosomes were captured in the anti CD-63 antibody modified gold electrode. Among these exosomes, only gastric cancer exosomes could trigger RCA to achieve the generation of large amount of G-quadruplex units. The products were then incubated with hemin to form hemin-G-quadruplex structures and catalysed H[2]O[2] system to produce electrochemical signal. The aptasensor was also validated in terms of the linearity and repeatability to demonstrate its potential in practice. Results: Anti-CD63, which can bind to the exosome surface marker was used as the capture probe. And the joint effects of hemin/G-quadruplex DNAzyme towards H[2]O[2] reduction and signal amplification produced by RCA reaction was used to generate significantly strong electrochemical and colorimetric response. Summary/Conclusion: In this work, we developed an electrochemical and colorimetric aptasensor for specific detection of gastric cancer exosomes. A specific gastric cancer exosome aptamer was selected and used as the detection probe. The aptasensor exhibits specificity towards target exosomes and high sensitivity. PT02: EVs in reproduction and pregnancy Chairs: Nanbert Zhong, Qi ChenLocation: Level 3, Hall A15:30–16:30 PT02.01 Placenta extracellular vesicles: a potential protective role against oxidative damage Qi Chen ^a, Chunlin Su^b and Larry Chamley^a ^aThe University of Auckland, Auckland, New Zealand; ^bFudan University of China, Shanghai, China (People’s Republic) Introduction: Extracellular vesicles (EVs) are lipid-enclosed packages of cellular contents including RNAs, protein and DNA that are produced by all eukaryotic cells to facilitate intercellular communication and regulation. Upon reaching their target cells, EVs may deliver their cargo and can induce signalling to alter the behaviour of target cells. During pregnancy, a large number of EVs are extruded from placenta (a foetal organ) into maternal circulation. Placental EVs are implicated in maternal immunosuppression and tissue repair. In this study we investigated whether placental EVs can prevent cell damage. Methods: EVs were isolated from first trimester placental explants (range from 8–12 weeks of gestation) and separated into micro- and nano-EVs by differential centrifugation. Human endometrium epithelial cells (HEE) were cultured for 18 h in the presence or absence of placental micro- or nano-EVs. After removal of excess EVs by washing with PBS, HEE cells were treated with 1 mM H[2]O[2] for 30 min and then the H[2]O[2] was removed by washing. The culture was continued for 18 h and proliferation of HEE was measured by Alamar Blue assay. The expression of H2AX, a marker of DNA damage in HEE cells was measured by IHC. Results: The proliferation of HEE was significantly reduced when HEE cells were treated with 1 mM H[2]O[2]. However this reduction of proliferation was significantly reversed by pre-treatment with either micro- or nano-placental EVs. In addition, the expression of H2AX was higher in HEE cells that had been treated with 1 mM H[2]O[2], but higher expression of H2AX was reduced in HEE cells that had been pre-treated with either micro- or nano-EVs. Summary/Conclusion: In this study, we found that pre-treatment with placental EVs can reduce the adverse effects of H[2]O[2] on HEE cell proliferation death and DNA damage. Our data suggest placental EVs have the ability to protective cells against oxidative damage. In pregnancy this property of placental EVs may assist the function of maternal cells that are exposed to increased oxidative stress. PT02.02 Maternal serum miRNA biomarkers for detection of placenta accreta Rebecca R. Adami ^a, Louise Laurent ^b, Victoria Fratto^c, Srimeenakshi Srinivasan^a, Cuong Tran^a, Peter DeHoff^a, Melissa Westermann^d, Allison O’Leary^e, Deborah Wing^d and Gladys Ramos^a ^aUCSD, San Diego, USA; ^bUCSD, La Jolla, USA; ^cNaval Medical Center, San Diego, USA; ^dUCI, Irvine, USA; ^eUCSF, San Francisco, USA Introduction: Failure to diagnose placenta accreta spectrum (PAS) prior to delivery is associated with worse outcomes. However, use of ultrasound and magnetic resonance imaging for this diagnosis is costly and imprecise. We hypothesize that levels of specific cell-free miRNAs in the maternal blood will differ among women with PAS, placenta previa and normal placentation. Methods: Women with suspected PAS, previa or normal placentation were prospectively recruited at three academic centres in the UC foetal Consortium. PAS was confirmed by pathologic evaluation. Maternal serum was collected antenatally, and total RNA was extracted, subjected to small RNA sequencing, and mapped to the miRBase human miRNA database. Groupwise differential expression analysis identified 13 candidate miRNAs, which were used to generate a support vector regression model for classification. The small RNA sequencing results for these candidate miRNAs were validated using qPCR. Results: 60 women were recruited: 18 PAS, 15 placenta previa and 27 normal placentation. The median gestational age at sample collection was 30w3d (IQR 28w–33w) and did not differ among groups (p = 0.13). The abundance of total miRNA reads as a percentage of all reads in the small RNA sequencing data was highest among women with PAS and lowest in normal placentation. Thirteen differentially expressed candidate miRNAs were identified. Support vector regression accurately classified samples into the three categories. Summary/Conclusion: The percent total miRNA was significantly higher in maternal serum in cases of PAS compared to normal placentation. Thirteen candidate miRNAs were differentially expressed among groups and were used in a training model to accurately classify samples. Our results suggest that maternal serum miRNAs have the potential to serve as biomarkers for accurate antenatal diagnosis of PAS. Studies in a larger independent cohort are needed for validation of these results. PT02.03 Effects of medium term storage on placental extracellular vesicles Larry Chamley, Julie Wang and Cherie Blenkiron The University of Auckland, Auckland, New Zealand Introduction: Studies on the function of isolated extracellular vesicles (EVs) are growing exponentially. However, as yet there is no consensus on how best to store EVs. We hereby conducted a term study to examine the stability of various cargos carried by placental EVs when stored at 4°C. Methods: First-trimester placental tissues were cultured for 24 h in medium supplemented with fluorescent cell tracker CMTPX (1 µg/mL). Debris was removed by centrifugation at 2000 ×g. Micro EVs were harvested by centrifugation at 20,000×g and subsequently nano-EVs were harvested following centrifugation at 200,000×g. The EVs were resuspended in PBS then aliquoted and stored at 4oC. CMTPX signal strength was examined by flow cytometry (AriaII) weekly. DNA was extracted, fortnightly, using Purelink Genomic DNA kit and measured using a Qubit dsDNA assay; and total proteins were isolated, fortnightly, with RIPA and quantified using BCA assay. Results: The proportions of micro and nano-EVs showing similar intensity of CMTPX signals did not change significantly for 3 months (n > 5) but an inconsistent and sample-dependent decline was observed thereafter. In contrast, the DNA content of EVs was stable for only 2 weeks. DNA quantities extracted from micro and nano-EVs declined by 40% and 60%, respectively, at week four compared to DNA extracted from freshly isolated EVs and thereafter remained stable until 8 weeks. Total protein in micro EVs was stable for 2 months. Whereas there was a 20% decline in the total protein extracted from nano-EVs by week 2 but levels remained stable thereafter. Finally, the corresponding placental tissues also stored at 4oC and processed in parallel showed continuous decline in both DNA and protein quantities. Summary/Conclusion: The CMPTX label incorporated into placental EVs may be stable for 3 months when stored at 4oC. However, the DNA of both micro and nano-EVs was less stable with a rapid decline upon storage. There was a marked difference in the stability of EV-associated protein with the protein content of nano-EVs being less stable than that of micro-EVs. Notably the total protein content of placental micro-EVs was remarkably stable when the EVs were stored at 4oC. Further work is required to assess the intactness/functionality of placental EVs after storage. Funding: Marsden Fund of the Royal Society of New Zealand PT02.04 Deciphering embryo-maternal communication; the dynamics of first contact between progenitor and progeny Kasun Godakumara ^a, Masoumeh Es-haghi^b, Keerthie Dissanayake^b, Freddy Lättekivi^b, Andres Salumets^c, Ülle Jaakma^d and Alireza Fazeli^b ^aDepartment of Pathophysiology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia; ^bDepartment of Pathophysiology, Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia; ^cCompetence Centre on Health Technologies, Tartu, Estonia; ^dInstitute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Tartu, Estonia Introduction: Failure of implantation has long been identified as a major challenge of assisted reproductive technologies. It is hypothesized that the embryo alters the endometrium to elevated receptivity by embryo-maternal cross talk. In previous communications, we have shown that RNA originating from JAr (analogue for trophoblast) cell line, packaged in extracellular vesicles (EVs) are transferred to RL95–2 (an analogue for endometrium) cell line and induce alterations in specific endometrial Zinc Finger Protein 81 (ZNF81) transcript. The objective of the current study was to test the hypothesis that only EVs from viable embryo alter ZNF81 transcript in the RL95-2 cell line. Methods: Human embryos were produced by classic in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). They were cultured individually for 20 h in Fert™ media (day 1), 48 h (day-3) in Cleav™ media and additionally 48 h in Blast™ media (day-5). At day-3, embryos with equal size blastomeres and no fragmentation were considered as normal. At day 5, embryos with identifiable inner cell mass, trophoblast and blastocyst cavity were considered normal while embryos forming mass of degrading cells were considered degraded. Conditioned media was collected from 6 normal day-3 embryos (three of which degraded by day 5), day-5 normal (n = 3) and degraded (n = 3) embryos, Cleav™ and Blast™ media. EVs were isolated using a sequential centrifugation and size-exclusion chromatography. A monolayer of RL95-2 cells (analogue for endometrium) was treated with isolated EVs. The change of gene expression of ZNF 81 and control genes (beta-actin, beta-2-microglobulin) in RL95-2 cells were measured using qPCR with absolute quantification. Results: Results exhibited that EVs derived both from day-5 normal blastocysts and day-3 embryos that undergo normal development significantly downregulated ZNF 81 expression in endometrial cells compared to untreated controls, cells treated with Cleave™ and Blast™ media EVs, cells treated with day-5 degraded embryos and day-3 embryos degrading on day-5 EVs. Control genes did not exhibit a significant change of expression. Summary/Conclusion: RL95-2 cells respond in different manners to EVs from normal and degraded human embryos. These findings can facilitate development of biomarkers for differentiating viable and degraded embryos at early stages after IVF. PT03: EV Nucleic Acid Biomarkers Chairs: Louise Laurent; Guoku HuLocation: Level 3, Hall A15:30–16:30 PT03.01 Circulating exosomal miRNAs as potential biomarkers for evaluation of preterm brain injury Kenta HT Cho^a, Bing Xu^b, Nina Zeng^b, Randall F. D’Souza^c, Cherie Blenkiron^d and Mhoyra Fraser ^b ^aDepartment of Physiology, Faculty of Medical and Health Sciences, The University of Auckland; ^bDepartment of Physiology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand; ^cDiscipline of Nutrition, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand; ^dThe University of Auckland, Auckland, New Zealand Introduction: Insults such as oxygen deprivation occurring in utero or during delivery have profound consequences on the neurological outcome of premature infants. This is a serious clinical problem, because treatment is a time-critical emergency and should be commenced within 6 h following injury. However, we simply do not know which preterm infants to treat due to the lack of sensitive biomarkers. Using our foetal sheep model of preterm brain injury, we sought to isolate exosomes from foetal plasma to establish whether they contain miRNA biomarkers that are associated with clinically significant neurologic outcomes. Methods: Chronically instrumented singleton foetal sheep at 0.7 gestation (term 145 days) received asphyxia induced by umbilical cord occlusion for 25 min. Size-exclusion chromatography (qEV) was performed for isolation and purification of extracellular vesicles (EVs) from plasma collected 4 h after occlusion from asphyxia (n = 10) and sham control (n = 10) foetuses. EV fractions were assessed for purity and quantity by nanoparticle tracking analysis and western blot against major EV protein markers. For biomarker identification, miRNA expression profiles from plasma EV fractions were determined by Affymetrix v4 microarrays. Results: Umbilical cord occlusion was associated with significant brain injury to areas commonly affected by asphyxia in preterm infants. Plasma EVs were characterised as rich in CD63 and HSP70, size ~100 nm, and with an exosome-like morphology by TEM. Profiling of EV-miRNAs revealed significant differences (log2 fold change > 2 or < −2 and p value < 0.05) between the asphyxia and sham control foetal groups. Strikingly, the majority of miRNAs differentially abundant with asphyxial-induced brain injury were less abundant, including miR-30b-5p, miR-30a-5p, miR-27a, let-7f, miR-223/3p, miR-221, miR-22-3p, miR-151–3p, miR-411–5p and miR-532 whereas only one miRNA (miR-455-3p) was more abundant. Summary/Conclusion: To the best of our knowledge, this study is the first to determine the usefulness of plasma exosomal miRNAs as biomarkers for the prediction of preterm brain injury. Our data reveal a unique plasma-derived exosomal miRNA profile, which may aid the early diagnosis of preterm brain injury. Funding: Neurological Foundation of New Zealand. PT03.04 Identification and Verification of Differentially Expressed MicroRNAs in the plasma microvesicles for the Diagnosis of moyamoya Disease Mi Jeong Oh ^a, Eun Hee Kim^a, Yeon Hee Cho^b, Dong Hee Kim^c, Ji Hee Sung^b, Eun Kyoung Shin^a and Oh Young Bang^d ^asamsung medical center, Seoul, Republic of Korea; ^bsamsung medical center, seoul, Republic of Korea; ^cSungkyunkwan University, seoul, Republic of Korea; ^dSamsung medical center, Seoul, Republic of Korea Introduction: There is no well-recognized miRNA biomarker for accurately predicting outcome in the presence of moyamoya disease (MMD), a unique cerebrovascular occlusive disease of unknown etiology^1,2. We performed a study of the significance of miRNAs expression in the plasma microvesicles (MVs) of MMD patients. Methods: The plasma MVs were purified from 38 healthy donors, 22 intracranial atherosclerotic stenosis (ICAS) patients and 40 moyamoya disease (MMD) patients. Plasma MVs were isolated using ultracentrifugation. We perfomed miR expression analysis using miRNome miScript miRNA PCR Array. Specific miRNAs were validated using real-time polymerase chain reaction, with normalization to an exogenous control (cel-miR-39). The angiogenic effects were measured by over-expressing or inhibiting specific miRNAs. Results: MiRNA profiles using miRNome miScript miRNA PCR array of three pooled plasma MV samples from patients with MMD, ICAS and controls revealed 222 differentially expressed serum miRNAs, including 115 upregulated and 107 downregulated miRNAs. In an independent MMD cohort, qRT-PCR confirmed that miR-A was significantly upregulated. Hsa-miR-A in the MMD group exhibited greater performance than ICAS group (AUC 0.735) in ROC curve analysis. To select target genes of specific miRNAs, we performed computational miR target prediction analysis (TargetScan) and found the seed sequence of CAV1 3’-UTR interacting with hsa-miR-A. The deregulation of miR-A by the transfection of HUVECs with pre-miR-A was significantly decreased tube formation of HUVECs. In addition, miR-A inhibited tube formation by suppressing the expression of CAV1at the posttranscriptional level, respectively, resulting in defective angiogenesis and MMD pathogenesis. Summary/Conclusion: Hsa-miR-A is markedly elevated in plasma MV of patients with MMD. The potential role of these microRNAs in the pathogenesis of MMD can contribute to find new diagnostic and therapeutic target of MMD. Funding: This study was supported by a grant from the Korean Healthcare Technology R&D Project, Ministry of Health & Welfare (HI17C1256) and Basic Science Research Program, the Ministry of Science, ICT and Future Planning (2018M3A9H1023675). PT03.05 Micro RNA- 451-5p in urinary exosomes for non-invasive monitoring of reno-protective response Manju Kumari, Aradhana Mohan, Ravi S. Singh, Rajni Sharma and Swasti Tiwari Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India Introduction: MicroRNA-451 in urinary exosomes (UE) had been demonstrated as a sensitive predictor of kidney injury in diabetic rats by us. Here, we determined whether miR-451 in UE could also assess severity of kidney injury and/or reno-protective response to therapy. Methods: Streptozotocin-induced (STZ, 50 mg/kg of body weight, i.p) type-1 diabetic rats were fed with normal chow diet (DM) or high cholesterol diet (DM + HCD), to increase the severity of nephropathy. Half rats in DM + HCD group were treated with atorvastatin (AT, 20 mg/kg body weight, DM + HCD + AT) for 8 weeks and rest remained untreated. After 8 weeks, urine was collected for exosomes-enrichment and albumin to creatinine ratio (ACR). Primary cultures of human proximal tubular cells (hPC) and their secreted exosomes were also studied. Taqman-based real-time qPCR was done for miR-451-5p. Results: At the end of the study, DM + HCD had significantly higher ACR while lower renal miR-451 levels, relative to DM and DM + HCD + AT rats. In addition, a strong negative correlation of renal miR-451 was found with ACR. In contrast to kidney tissue, UE analysis revealed a positive correlation of miR-451 levels with ACR in these rats. Moreover, miR-451 in UE was significantly higher DM + HCD rats relative to DM and DM + HCD + AT rats. In vitro studies confirmed the direct effect of hyperglycaemia / mechanical on miR-451 expression. Summary/Conclusion: Severity of kidney injury in rats is associated with lower renal miR-451 and higher UE miR-451. Statin attenuated renal injury and miR-451 levels in UE, while improving renal levels. MiR 451 analysis in UE may assess therapeutic response of reno-protective drugs non-invasively Funding: Funded by ICMR and DBT, Govt. of India. PT03.06 Circulating miR-451a is a useful biomarker for haemolysis Yukichi Takada, Tatsuki Shibuta and Tsukuru Umemura International University of Health and Welfare, Okawa City, Japan Introduction: Red blood cells (RBCs) are circulating enucleated cells, and its main component is haemoglobin carrying O[2]/CO[2]. RBCs contain erythroid lineage-specific microRNA (miRNA), miR-451a. Biogenesis of miR-451a depends on the activity of Ago2, and also circulating miR-451a is mainly Ago2-bound, non-exosome miRNA. For utilizing circulating miR-451a for diagnosis of anaemia which is caused by destruction of RBCs (haemolysis), it is important to evaluate distribution pattern of circulating miR-451a. Methods: We analysed 120 remnant serum samples obtained from routine blood drawing for laboratory testing. The study was done under permission of The Ethical Committies of International University of Health and Welfare and Kohoukai Takagi Hospital. Serum miRNAs were analysed using TaqMan microRNA assay kits and RT-qPCR (ABI 7500fast). Exosomes were obtained using the ultracentrifugation method and the precipitation method. Results: We analysed 120 remnant serum samples obtained from routine blood. The sensitive Hb detection method using 414 nm absorbance (NanoDrop2000, ThermoFischer) showed that 100% of blood sampling had haemolysis. The levels of serum miR-451a also increased in all samples. Ultracentrifugation method and precipitation method showed more than 90% of serum miR-451a is in the non-exosome fraction. Summary/Conclusion: miR-451 is a unique miRNA which is in the non-exosome fraction. The measurement of serum miR-451a is a sensitive and specific biomarker for haemolysis. Funding: This work was supported in part by a grant from the Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number: JP17K09020). PT03.07 Circumventing qPCR inhibition to improve amplification of exosomal miRNAs in preterm foetal sheep heparinised plasma Bing Xu ^a, Mhoyra Fraser ^1 and Kenta HT Cho ^b ^aDepartment of Physiology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand; ^bDepartment of Physiology, Faculty of Medical and Health Sciences, The University of Auckland Introduction: Exosomal miRNAs have been identified in plasma, which has led to an interest in their potential as biomarkers of neural injury responses in survivors of prematurity. The chronically catheterized preterm foetal sheep model is a uniquely versatile model to advance our understanding of the pathophysiological mechanisms underlying preterm brain injury and develop new therapeutic strategies. However, to ensure patency of catheters implanted into foetal vessels heparinised saline is infused continually. Heparin can confound detection of plasma miRNAs. Here we present an optimal procedure to collect and detect exosomal miRNAs in foetal plasma that improves sensitivity and performance of qRT-PCR. Methods: Size-exclusion chromatography (SEC; qEV) and commercial ExoRNeasy were performed for isolation and purification of extracellular vesicles (EVs) on foetal plasma collected in K2EDTA tubes in which varying amounts of heparin or enoxaparin (0–160 IU/mL) had been added post-thawing. RNA was extracted from qEV fractions (miRNeasy), ExoRNeasy EVs, or from whole plasma (miRNeasy) and treated with or without heparinase I to remove contaminating heparin. Levels of endogenous miR-16 and let-7a were measured using qRT-PCR. Results: Important differences in EV-miRNA abundance were observed between isolation methods. Heparinase I treatment improved detectability of the miRNAs in a dose-dependent manner in heparin/non-heparin spiked whole plasma and when isolated using the ExoRNeasy kit. Strikingly, SEC removed endogenous heparin from non-heparin spiked plasma collected from catheters infused with low-dose heparin and this effect was not improved by heparinise I. Further, SEC partially removed contaminating effects of heparin in heparin spiked whole plasma. Summary/Conclusion: Treatment of foetal sheep plasma with heparinase I enables utilisation of qRT-PCR for reliable miRNA quantification. SEC also enables removal of inhibitory heparin and therefore should be considered as a standard step for isolation and detection of EV-miRNAs in foetal plasma. Funding: Neurological Foundation of New Zealand. PT03.09 A particle-based multiplex RT-qPCR for measuring circadian rhythm-associated genes Kim Mi Yeon ^a, Jung Seungwon^a, Kim Junsun^a, Lee Heon Jung^b and Kim Sangkyung^a ^aMolecular Recognition Research Center, Materials and Life Science Research Division, KIST, Seoul, Republic of Korea; ^bSleep-Wake Disorders Center, Korea University Anam Hospital, Seoul, Republic of Korea Introduction: Biological clocks which regulate the expression of genes related to the circadian metabolism of living organisms are directly linked to human health and disease. Many studies also have suggested to analyse mental health issues such as depression and bipolar disorder through molecular analysis of circadian rhythm-associated genes recently. These genes are generally analysed by reverse-transcription real-time PCR (RT-qPCR) but conventional methods require considerable time, cost and amount of the sample to figure out multiple genes at the same time. Methods: We introduce a sensitive and multiplex RT-qPCR using hydrogel particles. One of the primer pair used in RT which captures and reverse-transcribes the target RNA is immobilized in the particle. The other primer is stored not to react during RT and released when amplification begins. This strategy can help the RT process to avoid non-specific products as well as the amplification to occur effectively. Results: In order to see RT-qPCR efficiency, PER3 RNA which is synthesized via in vitro transcription (IVT) of complementary DNA, and total RNA extracted from LNCAP cell were tested with serial dilution. As a result, they showed high amplification efficiency of 95% and 98%, respectively. In addition, the expression level of genes was successfully measured even from a single cell. Expression pattern of 8 circadian rhythm-associated genes was acquired from total RNA of circadian rhythm-synchronized HeLa cells. In addition, exosomal RNAs were monitored same cells every four hours for 48 h in order to obtain molecular circadian rhythm cycles. Each gene showed unique pattern of expression. Summary/Conclusion: For non-invasive diagnosis, clinical sample of human epithelial cells, saliva exosome and hair cells were confirmed particular patterns. Further study will emphasize to take a snapshot of molecular circadian rhythm through the combination of expression levels of associated genes measured at a certain time point for diagnosis of the mood disorder. PT04: EV Nucleic Acid Cancer Biomarkers Chairs: Christian Preußer; Harry HolthoferLocation: Level 3, Hall A15:30–16:30 PT04.01 Unveiling of extracellular exosomal miRNA profiles of breast cancer Shang-Che Kuo ^a, Ko-Chien Chen^a, Takahiro Ochiya^b, Wen-Hung Kuo^c, King-Jen Chang^d, Kuo-Kan Liang^e and Tang-Long Shen^a ^aNational Taiwan University, Taipei, Taiwan (Republic of China); ^bDepartment of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Shinjyuku-ku, Japan; ^cNational Taiwan University Hospital, Taipei, Taiwan (Republic of China); ^dTaiwan Adventist Hospital, Taipei, Taiwan (Republic of China); ^eAcademia Sinica, Taipei, Taiwan (Republic of China) Introduction: In an era of precision medicine, biomarker discovery is indispensable for early detection, therapeutic efficacy monitoring and outcome prediction. MicroRNAs within patient serum exosome have emerged as significantly measurable biomarkers, which abundantly existed in the form of liquid biopsies, for several diseases, including cancers. They are essential regulators of global mRNA expression in cells. Aberrant regulation of miRNA can enables resulting in tumour initiation, drug resistance and metastasis in cancer. miRNA assays are convenient for large-scale studies covering multiple miRNA targets and realistic in screening across diverse breast cancer types for early detection or factors that drive cancer progression. Methods: In this study, we collected patient serum samples from four major molecular subtypes: luminal A, luminal B, HER2+ and triple negative types, and breast cancer patients with benign tumour and ductal carcinoma in situ (DCIS). Microarray analysis of miRNA expression was utilized and unique serum miRNA signatures between non-cancer (including benign, DICS) and breast cancer patients were identified with differential expression analysis and the common features selection method – elastic net. All combinations of selected miRNAs were modelled with three different approaches, including generalized linear model (GLM), linear discriminant analysis (LDA) and support vector machine (SVM). Results: To analyse more generally, we applied various normalization methods and got different outcomes after feature selection. All the selections were used to fit prediction models. After applying the filter criteria based on accuracy evaluation by 10-fold cross-validation, the top selected miRNAs showed the consistency of different prediction methods, and the union of these selections was used in the further modelling. The results confirmed that few miRNAs are enough to implement early detection at high accuracy. Summary/Conclusion: Through identifying these heterogeneous compositions of the cancer cells, understanding of the molecular mechanisms underlying these identified biomarkers, which is essential in developing effective treatments and translational research, could be established. PT04.03 Hypoxia may promote tumour aggressiveness and extracellular vesicle-mediated cell-to-cell communication in multiple myeloma Kyung Ju Ryu ^a, Ji Young Lee^b, Sung Won Lim^b, Chaehwa Park^c, Kihyun Kim^d and Seok Jin Kim^c ^aSungkyunkwan University, Seoul, Republic of Korea; ^bSamsung Medical Center, Seoul, Republic of Korea; ^cSungkyunkwan University, Samsung Medical Center, Seoul, Republic of Korea; ^dSungkyunkwan University, Samsung Medical Center, Seoul, Republic of Korea Introduction: Hypoxia is one of important features of tumour microenvironment, and tumour cells under hypoxia can acquire aggressive characteristics including drug resistance. Thus, tumour progression and resistance to therapy are associated with hypoxic tumour microenvironment. Multiple myeloma (MM) is a neoplasm of bone marrow plasma cells. Bone marrow is hypoxic compared to other organs, thus, tumour aggressiveness of MM could be closely associated with hypoxic microenvironment. Extracellular vesicle is a small vesicles containing a wide range of functional proteins, mRNAs and miRNAs that are actively secreted via exocytosis. In this study, we investigated the effect of hypoxia on the EV formation of MM cells to identify the underlying mechanism of tumour aggressiveness in MM cells. Methods: We conducted a long-term culture of MM cell lines under hypoxic conditions (1% and 2% of O2 for 4 weeks), and compared with same MM cell lines cultured under normal oxygen concentration (20%). The RNA expression profiles of MM cells under hypoxia were also compared with that of cultured cells under normal oxygen condition. The EV derived from MM cells was isolated using ExoQuick‐TC solution and assessed by transmission electron microscopy, Nanoparticle tracking analysis and Western blot. Results: The overexpression of HIF-1α was demonstrated in MM cells under long-term hypoxia, and the expression of stem cell markers were more increased in MM cells under hypoxic condition compared to normal oxygen concentration The RNA sequencing showed up-regulation of gene associated with production of EV in hypoxic cultured cells. When we measured EV from hypoxic cultured MM cells, the amount of EV was significantly higher in hypoxic MM cells than normoxic control group. To identify specific alterations associated with hypoxic MM cells, we profiled miRNAs derived from EV of hypoxic MM cell lines and those of normoxic MM cell lines. These results identified eight miRNAs with significantly different expression between MM cells – derived EV. Summary/Conclusion: We demonstrated the characteristics of long-term hypoxic MM cell-derived EV. The EV-mediated cell-to-cell communication under hypoxia might be associated with the content of miRNA in MM cell-derived EV, and it might influence tumour aggressiveness of MM cells. PT04.04 Deep sequencing identified serum exosomal miR-181a-5p as an indicator for bone-metastatic prostate cancer Yanqing Wang ^a, Yu-Xiang Fang^b, Baijun Dong^a, Wei-Qiang Gao^b and Wei Xue^a ^aDepartment of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China (People’s Republic); ^bState Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China (People’s Republic) Introduction: Prostate cancer (PCa) is the most common male malignancy worldwide with high heterogeneity from tumorigenesis to metastasis. Although bone metastasis is the most critical metastatic event, at present, there has been no specific and accurate biomarker for its diagnosis or differentiation at an early stage of PCa. Given the fact that the profiling change of exosomal miRNAs can work as a biomaker for metastasis in multiple tumours, we seek to identify exosomal miRNAs in patient’s serum as indicators for bone-metastatic PCa. Methods: The profiling change of serum exosomal miRNAs in patients with either benign prostatic hyperplasia (BPH) or localized or bone-metastatic PCa was detected by miRNA-seq and miRNA-chip array, respectively. Prospective miRNAs were further confirmed using TaqMan miRNA assay in two independent validation cohorts of total 127 patients with either BPH or localised or bone-metastasic PCa. Logistic regression analysis was performed to evaluate the diagnostic association of candidates with bone metastasis. Accuracy estimate of each candidate for the diagnosis of bone-metastatic PCa was quantified using the area under the receiver-operating characteristic curve (AUC). Results: By miRNA-seq and miRNA-chip array, we found four prospective exosomal miRNAs including miR-181a-5p with significant differences between localized and bone-metastatic PCa groups (p＜0.05, fold change ≥1.5 or ≤0.5). In the validation cohorts, logistic regression analyses indicated that miR-181a-5p and miR-320a were significantly associated with bone-metastatic PCa. The AUC analyses identified miR-181a-5p as the best biomarker with the AUCs 93.1% for diagnosis of PCa and 73.9% for that of tumour bone metastasis. Summary/Conclusion: Serum exosomal miR-181a-5p is a promising diagnostic biomarker for bone-metastatic PCa. Further validation is needed. Funding: National Natural Science Foundation of China (81630073 to W-QG, 81874097 to Y-XF, 81672850 to BD, 81572536 and 81772742 to WX) PT04.05 Exosomal miRNAs and proteins signature as prognostic biomarkers for early stage epithelial ovarian cancer Shayna Sharma ^a, Andrew Lai^a, Dominic Guanzon^b, Terry Morgan^c, Lewis Perrin^d, John Hooper^d and Carlos Salomon^b ^aExosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane, Australia; ^bExosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane, Australia; ^cDepartment of Pathology and Obstetrics, Oregon Health and Science University, Portland, OR, USA; ^dMater Health Services, South Brisbane, QLD, Australia, Brisbane, Australia Introduction: Epithelial Ovarian Cancer (EOC) is the leading gynaecological malignancy worldwide due to the limitations of current detection tests. The 5-year survival rate with early detection is 90% compared to 20% with late detection. Unfortunately, only 30% of the cases are detected early. Thus, it is essential to develop a novel and minimally invasive method to identify patients at an early stage. Exosomes have shown promise as biomarkers as they encapsulate vital information. Therefore, the aims of this study were to (i) determine the content of circulating exosomes at early stages of EOC, and (ii) to determine the prognostic performance of an early-ovarian cancer screening test to identify women at risk of developing EOC. Methods: Exosomes were isolated from the plasma of patients with either benign disease (n = 50) or Stage I/II EOC (n = 28), through differential centrifugation and size exclusion chromatography. Exosomes were characterized using Nanoparticle Tracking Analysis, Western Blot and Electron Microscopy. Exosomal proteins were profiled using Liquid Chromatography–Mass Spectrometry (LC-MS/MS) and SWATH analysis. An Illumina TrueSeq Small RNA Library Prep kit was used for exosomal miRNA profiling. A binomial classification algorithm was generated using a boosted logistic regression analysis (WEKA machine learning software (ver 3.6.12)) of the results obtained from the benign and Stage I/II samples. The algorithm was built using 5 miRNAs and 5 proteins identified through circulating exosome profiling. The expression of specific miRNAs was confirmed using RT-qPCR to validate the miRNA sequencing results. Results: miRNAs and proteins were identified as being differentially expressed across EOC progression. The algorithm that we built delivered discrimination between women with EOC (Stage I/II) compared to benign. The classification efficiency was assessed by ROC curve analysis (area under the curve (AUC) was 0.785 ± 0.091 (p = 0.0106)) with positive and negative predictive values of 75% and 76%, respectively. Summary/Conclusion: We propose that the combined measurement of exosomal miRNAs and proteins might allow for the early identification of women with EOC, distinguishing between patients with benign disease and patients with Stage I/II EOC. Future directions involve the validation of the proposed miRNAs and proteins in a larger cohort. Funding: OCRF. PT04.06 Circulating Extracellular vesicle (EV)-encapsulated microRNAs as a biomarker of breast cancer Clodagh O’Neill^a, Róisín Dwyer ^b, Sonja Khan^a, Katie E. Gilligan^c and Peter Dockery^b ^aNational University of Ireland, Galway, Galway, Ireland; ^bNUI Galway, Galway, Ireland; ^3National University of Ireland Galway, Galway, Ireland Introduction: Early detection of breast cancer is the key to improve patient survival. There is currently no robust biomarker available to detect breast cancer. Extracellular vesicle encapsulated microRNAs (EV-miR) provide novel potential in this field. EVs are tiny nanoparticles released by all cells in the body that contain various bioactives including miRNA, believed to reflect the characteristics of the parent cell. Evidence suggests that tumour associated EVs have a distinct miRNA expression profile from normal cells, and could hold novel biomarker potential. Methods: This study aimed to determine the circulating EV-miR profile of tumour-bearing animals compared to healthy controls, and relate this to the EV-miR profile secreted by tumour cells in vitro. EVs were isolated from the supernatant of HCC-1954 breast cancer cells expressing luciferase (HCC-luc). EVs were also harvested from healthy BALB/c nude mice or those bearing mammary fat pad HCC-luc tumours. Serum and media EVs were isolated by differential centrifugation, followed by microfiltration and ultracentrifugation. EVs were characterised by Nanoparticle Tracking Analysis (NTA), western blot and Transmission Electron Microscopy (TEM). Next-Generation Sequencing (NGS) targeting miRNA was preformed to compare the HCC-luc EV-miR profile in vitro and in vivo. Results: EVs were successfully isolated and demonstrated to express CD63, CD9 and CD81. Using NTA, size distribution was confirmed to be of the predicted range of 30–120 nm. A range of miRNA was detected in the HCC-luc EVs in vitro. Interestingly, nine of these miRNAs were present at significantly higher levels in the EVs than the cells from which they were released, e.g. miR-184 and let-7c. Initial analysis revealed that a number of miRNAs packaged into EVs by HCC-luc cells In Vitro were also detectable in circulating EVs isolated from tumour-bearing animals. Summary/Conclusion: EVs are thought to represent a fingerprint of the cell from which they are released, and hold great potential as biomarkers for breast cancer detection. Further understanding of miRNA trafficking and transfer in EVs will shed light on their true potential in the cancer biomarker and therapeutic setting. Funding: I am funded by the National Breast Cancer Research Institute (NBCRI). PT04.07 Role of exosomal microRNA as a biomarker for extranodal NK/T-cell lymphoma Kyung Ju Ryu ^a, Chaehwa Park^b, Won Seog Kim^b and Seok Jin Kim^b ^aSungkyunkwan University, Seoul, Republic of Korea; ^bSungkyunkwan University, Samsung Medical Center, Seoul, Republic of Korea Introduction: Extranodal natural killer (NK)/T-cell lymphoma (ENKTL) is one of aggressive subtype of non-Hodgkin lymphoma, and all tumour cells are inarguably infected with Epstein–Barr virus. The cell to cell interaction and association with tumour microenvironment could be important for this disease entity. Exosomes are small membrane vesicles of 30–150-nm diameter that plays an important role in the tumour microenvironment, and they are actively secreted by most cell types, including cancer cells. In particular, the intra-exosomal microRNA is known as being important for intercellular communications. However, the clinical significance of exosomal miRNAs in ENKTL has not been examined. Thus, we characterized exosomal miRNAs in ENKTL and analysed their effect on the outcomes of patients. Methods: We isolated exosomes from ENKTL patient serum and lymphoma cell lines using ExoQuick and analysed by transmission electron microscopy, Nanoparticle tracking analysis (NTA) and Western blot. We performed exosomal microRNA profiling via the nCounter miRNA expression assay on exosomes from 45 ENKTL patients and lymphoma cell lines. Results: We isolated and characterized exosomes from NKTL patient serum and cell lines using ExoQuick, and analysed by TEM, NTA and Western blot. The serum-derived exosomes had a diameter of 95.84 ± 11.37 nm and exosome concentrations ranged from 0.25 to 14 × 1012/mL. We verified exosomes morphology and size using TEM, and detected exosomal markers, including Alix, and CD63 by western bolt. We performed miRNA microarrays to compare exosomal miRNAs of patients with ENKTL having good and bad prognosis. As shown in the microarray results, we found various miRNAs that were differentially contained in the serum – derived exosomes of NKTL poor relative to good subjects. These results identified 30 miRNAs with significantly different expression between NKTL samples. Five of these miRNAs were up-regulated and 25 ware down-regulated in the serum-derived exosomes of NKTL bad compared to the good subjects (p value < 0. 05). We identified two exosomal miRNA signatures, has-miR-320e and miR-4516, that were associated with poor outcomes with regard to OS and PFS. Summary/Conclusion: Our study provides that exosomal miRNA, miR-320e and miR-4516, may serve as potential diagnostic and prognostic biomarker in NKTL. PT04.08 Cancer-derived exosomes enriched from patient plasma strongly mirror parent tumour and enable subtyping of early stage breast cancer via liquid biopsy Christine Coticchia ^a, Robert Kitchen^b, Sudipto Chakrabortty^b, Douglas Roberts^a, Lisa Bedford^c, Sunita Badola^c, Sylvie Vincent^c, Seth Yu^B and Johan Skog^d ^aExosome Diagnostics, Waltham, USA; ^bExosome Diagnostics, Inc, Waltham, USA; ^cTakeda, Cambridge, USA; ^dExosome Diagnostics, Inc., Waltham, MA, USA Introduction: Tumour-derived molecular signatures of breast cancer (BCa) have accelerated personalized medicine as prognostic and predictive indicators leading to improved clinical outcomes. Currently, molecular profiling is performed on biopsied breast tumour tissue but our goal of “liquid biopsy” is to obtain disease-relevant genetic material non-invasively by capturing exosomes, cfDNA, or protein from bodily fluids. Unfortunately, a major limitation of liquid biopsy stems from the scarcity of disease-relevant material compared to background. Here we describe an enrichment process in plasma capable of isolating cancer specific exosomal subpopulations originating from early stage breast tumours. Methods: Tumour-specific surface markers on exosomes were targeted and enriched from plasma obtained from stage I/II ER positive / HER2 negative BCa patients and age-matched controls. RNA-sequencing was performed on total RNA isolated from 15 BCa tumour tissues (FFPE) and 15 patient-matched plasma exosome samples (with and without exosome enrichment). We also sequenced RNA from 12 healthy breast tissues (FFPE) and plasma exosomes from 10 healthy post-menopausal women (with and without tumour exosome enrichment). RNA-seq data were used for gene-level differential abundance analysis. Results: Tumour-derived exosome enrichment was observed in 63% of the BCa patients with detectible levels of the target antigens in their plasma. RNA-seq gene expression profiles of these enriched exosomes were highly correlated with those of the breast tumour FFPE samples. Tumour-enriched exosomal RNA abundance clustered most tightly with the FFPE tissue derived from the same patient; even more so than BCa FFPE samples correlated to each other. The strength of the correlation between BCa enriched plasma exosomes and matched patient tissue was sufficient to enable correct tumour subtyping (by both PAM50 & IntClust gene targets) using only the enriched plasma exosomal RNA. Summary/Conclusion: Tumour-specific exosome enrichment improved plasma-derived exosomal RNA signal to noise and revealed RNA profiles that closely reflect the donor tumour, thus enabling the detection and characterization of early stage breast cancers. PT04.09 Exosomes: the same team for hepatocellular carcinoma development on the background of HCV and ergotism? Alisa Petkevich, Alexandr Abramov, Mohamed Kadle and Vadim Pospelov Peoples’ Friendship University of Russia (RUDN University), Moscow, Russia Introduction: Hepatocellular carcinoma (HCC) may be caused by a wide variety of reasons, two possible of them are hepatitis C virus infection (HCV) and alkaloids contained in the ergot (Claviceps). Anyway, not all of the individuals infected with HCV or living in regions endemic for ergot develop HCC so it is reasonable to develop biomarker panel for identification of risk groups for HCC. Exosomes seem to be an ideal source of such biomarkers as far as they contain exactly the information molecules packed by cells during its physiological (or pathological) functioning. Methods: 48 plasmas of patients with HCC from Somalia (from a region with a high degree of ergot alkaloides in food), and 18 plasmas of HCC (Russia) on the background of cirrhosis due to HCV. Exosomes were isolated from plasma by differential ultracentrifugation following free-flow electrophoresis. MiRNA let-7a-5p, −224-5p, −106b-3p, −126-5p, −122-5p, −16-5p and −34a-5p were determined in exosomes by qPCR-RT. Same free miRNA from plasma were determined. PD-L1 expression was assessed on the surface of exosomes by TEM and HR-FCM. PD-L1 expression was also assessed on the surface of exosomes isolated from plasma of healthy donors (n = 8). Results: There was a slight difference in exosomal miRNA profile of plasma from HCC on the background of HCV and on the background of HCV and living in ergot region. PD-L1 expression on the surface of exosomes from HCC plasmas were higher (MV 35,8% for both HCC groups, MV 5% for healthy donors group). Plasma free miRNA profiles were different inside every HCC group. Summary/Conclusion: According to our results, exosomal miRNA identification in HCC patients seem to be more accurate than plasma free miRNAs, further research is needed in order to identify whether it is reasonable to use both free and exosomal miRNAs. The difference in miRNA profiles of HCC patients on the background of HCV or alkaloids of ergot may allow supposing different epigenetics dysregulation happen in HCC depending on the trigger factor. PT04.10 Exosomal sorting of circRNA promotes cancer progression and serves as a novel biomarker for gastric cancer Rong Li ^a, Junyi Wang^b, Xu Zhang^b, Hui Qian^c and Wen Rong Xu^d ^aJiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of Medicine, Jiangsu University, Zhenjiang, China, Zhenjiang, China (People’s Republic); ^bZhenjiang Key Laboratory of High Technology Research on Exosomes Foundation and Transformation Application, Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of Medicine, Jiangsu University, Zhenjiang, China (People’s Republic); ^cZhenjiang, China (People’s Republic); ^dZhenjiang Key Laboratory of High Technology Research on Exosomes Foundation and Transformation Application, Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of Medicine, Jiangsu University, ZhenJiang, China (People’s Republic) Introduction: Exosomes are critical mediators of intercellular communication and promising biomarker for cancer. However, whether the release of exosomes has an effect on donor cells has not been well investigated. In this study, we aimed to identify the clinical values of exosomal circRNAs in gastric cancer (GC). Meanwhile, we explored the biological roles and mechanisms during GC cells selectively sorting exosomal circRNAs. Methods: Database circ2Traits and starBase v2.0 were used to screen potential GC related circRNAs and validated their expression levels in over 50 paired serum or exosomes from GC patients and healthy volunteers, or 100 cancer tissues and adjacent normal tissues from GC patients. Receiver operating characteristic curve, clinicopathological features analysis and overall survival (OS) and disease-free survival (DFS) curve were made to evaluate the clinical relevance of these circRNAs and select circ1477 as potential biomarker for further studies. Circ1477 overexpression and knockdown experiments were conducted to assess the effects on GC progression in vitro and in vivo. RIP, luciferase assay, RNA FISH and rescue experiments were applied to demonstrate its molecular mechanism. Results: We found that the level of circ1477 in serum or serum exosomes of GC patients was significantly higher than that in healthy volunteers. Whereas, the level of circ1477 in cancer tissues and was remarkably lower compared to adjacent normal tissues of GC patients, which was associated with lymph node metastasis and prognosis. Also, the expression of circ1477 was observed to be decreased in GC cell lines while increased in their derived exosomes, suggesting that circ1477 could be selectively sorted into exosomes by GC cells. Furthermore, cytoplasmic circ1477 could suppress the migration and invasion of GC cells acting as miRNA sponges while knockdown of it could reverse these effects. Summary/Conclusion: Taken together, our findings indicate that tumour suppressive circ1477 could be selectively sorted into exosomes to promote tumour progression and serve as a potential biomarker for gastric cancer. Funding: National Natural Science Foundation of China: (81572075); Technology Development Project of Jiangsu University (20180361) PT04.11 Development of non-invasive tests for prostate cancer David J. Whittaker ^a, Bianca Dobson^a, Clare Elton^a, Damian White^a, Nancy Frédérickx^a, Nicola Jackson^a, Niha Phukan^a, Mike Herbert^a, Rebecca Girvan^a, Lia Bootten^a, Genevieve Johnston^b, Dug Yeo Han^a, Ben Curran^a, Jennifer Barnes^a, Robert Mitchell^a and Keith Hudson^b ^aCaldera Health, Auckland, New Zealand; ^bCaldera Health, Auckland, USA Introduction: Prostate cancer (PC) is the most common non-skin cancer in males and is fast becoming the most frequently diagnosed cancer in men. Despite significant advances in diagnosis and treatment PC remains a leading cause of cancer deaths. Analysis of PSA in blood has long been used for early diagnosis and monitoring but is flawed by low sensitivity and a high rate of false positives, with negative health consequences including the overtreatment of many indolent prostate cancer tumours. Caldera Health is developing non-invasive liquid biopsy tests for prostate cancer to improve upon and replace the controversial serum PSA test. Methods: Through a series of clinical studies, Caldera Health has identified promising RNA biomarkers for PC diagnosis. Preliminary experiments indicated that in urine a far greater proportion of prostate RNA is localised in extracellular vesicles (EVs) than in cellular material. A simple and reliable process was optimised to concentrate urinary EVs and a novel method was developed to specifically isolate the EV’s of prostatic origin with high efficiency. Subsequently a clinical study was performed using qRT-PCR to quantify RNA biomarkers in approximately 300 urine samples collected from men scheduled for prostate biopsy tests. The clinical study participants provided informed consent and the study was approved by recognised medical ethics committees in New Zealand and Australia. Results: Comparison of the qPCR data for prostate, bladder and kidney-specific genes indicated our prostate vesicle isolation method successfully reduces contamination with vesicles from both kidney and bladder. The clinical study data was used to develop accurate prostate cancer diagnostic models. Summary/Conclusion: Caldera Health has identified EV RNA biomarkers associated with prostate cancer and developed a novel method to specifically isolate prostate-derived EVs from urine. We have tested multiple biomarkers and developed gene signatures identifying prostate cancer with high sensitivity and specificity. PT05: EV Biogenesis Chairs: Imre Mager, Hollis ClineLocation: Level 3, Hall A15:30–16:30 PT05.01 Uncovering the role of heparan sulphate proteoglycans in extracellular vesicle biogenesis: potential tools for improved therapies Rebecca L. Morgan ^a, Rebecca Holley^b, Jason Webber^c, David Onion^d, Cathy Merry^d and Oksana Kehoe^e ^aKeele University, Nottingham, UK; ^bThe University of Manchester, Manchester, UK; ^cCardiff University, Cardiff, UK; ^dUniversity of Nottingham, Nottingham, UK; ^dKeele University, Oswestry, UK Introduction: Many cell types deliver therapeutic effects by secreting extracellular vesicles (EVs). Therefore, EVs could be used as an alternative approach to cell-based therapies, overcoming many cell-associated challenges. EVs could be optimised to generate potent therapies through manipulating the mechanisms driving EV biogenesis. We aim to prove this concept by altering the heparan sulphate (HS) chains found on syndecan, a key component in the syndecan-syntenin-ALIX mechanism. We predict that HS is involved in cargo selection due to its ability to form interactions with a wide range of factors. In addition, the structure of HS influences the activity of heparanase, a regulator in the rate of EV production. Therefore, structural alterations to HS could allow the cargo (thus therapeutic activity) to be modulated whilst simultaneously increasing EV yields. Methods: MCF-7s mutated to alter expression of HS biosynthetic enzymes were generated using CRISPR-Cas9. Wild type and mutant MCF-7s were cultured in bioreactors using media containing EV-depleted Knockout Serum Replacement. EVs were isolated by differential ultracentrifugation and characterised using Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA) and Western Blot. Results: A FACS-based method has been developed to characterise and sort EVs based on their displayed HS. The cargo and functional activity of the sorted populations was then assessed. Since heparanase influences EV production rates, MCF-7s were incubated with a heparanase inhibitor (OGT2115). Subsequent alterations to soluble, cellular and vesicular HS composition was analysed by fluorescent labelling and SAX-HPLC identification. EV size and concentration was assessed using TEM and NTA. Summary/Conclusion: Optimising EVs may generate highly efficacious and cost-effective treatments in comparison to those based on the producer cell line. Alterations to the HS structures on syndecan could be an ideal method for optimisation. Funding: This PhD project is funded by EPSRC and MRC. PT05.02 Augmentation by GnRH of ectosome containing annexin A5 formation by blebbing of pituitary gonadotropes and its biological effect Mitsumori Kawa “a” minami ^a, Fungbun Numfa^b, Makoto Sugiyama^c, Ryota Terashima^d and Shiro Kurusu^e ^aVeterinary Physiology, Faculty of veterinary medicine, Okayama University of Science, Imabari, Ehime, Japan; ^bKhon Kaen University, Towada, Japan; ^cKitasato University, Towada, Japan; ^dVeterinary Physiology, Kitasato University, Towada, Japan; ^eVeterinary Physiology, Kitasato University, Towada, Japan Introduction: We have demonstrated that gonadotropin releasing hormone (GnRH) stimulates the synthesis of annexin A5 (ANXA5), a member of annexin family protein, in the pituitary gonadotropes and ANXA5 augments GnRH stimulation of gonadotropin secretion. It is, however, obscure how ANXA5 augments gonadotropin release at gonadotropes. As ANXA5 was demonstrated both in and out of cells, in the present study, we examined translocation of ANXA5 in response to GnRH stimulation in relation to the release of luteinizing hormone (LH). Methods: Rat pituitary tissues, primary pituitary cells and LβT2 gonadotrope cells were used. The conditioned medium was sequentially centrifuged at 20,000 ×g and 110,000 ×g to obtain ectosome and exosome respectively. Immunochemistry for ANXA5 and LHβ were performed. Transmission electron-microscope (TEM) was also used. Results: GnRH agonist (GnRHa) administration showed the formation of blebs containing ANXA5 on LβT2 cells and primary pituitary cells after only 10 and 30 min incubation. Hemi-pituitary gland was cultured with GnRHa and TEM showed that the boundary of GnRHa stimulated gonadotrope-like cell became obscure with many bubble like particles after 30 min incubation. The 20,000 ×g and 110,000 ×g particles were increased by the GnRHa treatment. ANXA5 was detected dominantly in 20,000 ×g pellet after treatment with GnRHa. It increased until 180 min. ANXA5 in 110,000 ×g pellet was also shown at 180 min. GnRHa treated 20,000 ×g particulate fraction significantly stimulated LH release in a dose dependent manner. Extracellular vesicle fraction prepared from plasma of one-week ovariectomized rats, in which GnRH secretion was expected to be augmented, showed significant increase of ANXA5 in the 20,000 ×g pellet. The blebbing induced by GnRH was inhibited by H89, protein kinase A inhibitor. It is suggested that Gαs signalling is necessary for GnRH stimulation of blebbing. Summary/Conclusion: Present study clearly demonstrates a hormonal regulation of ectosome formation and a novel mechanism of cell–cell communication by means of ANXA5 including ectosome. PT05.03 Investigation into a novel role for the prolyl isomerase cyclophilin A during Extracellular vesicle signaling in cancer Yunjie Wu ^a, Kieran Brennan^b and Margaret M. Mc Gee^a ^aUCD School of Biomolecular & Biomedical Science, Conway Institute, University College Dublin, Dublin, Ireland; ^bUniversity College Dublin, Ireland Introduction: Extracellular vesicles (EVs) released from cells mediate local and systemic cell–cell communication via the horizontal transfer of functional protein, DNA and RNA into recipient cells. Evidence reveals that tumour-derived EVs mediated intercellular communication between tumour cells and normal cells within the tumour microenvironment to initiate metastatic niche formation. Thus, disruption of EV-mediated tumour-niche interactions is a novel strategy for metastasis prevention. However, significant challenges in EV biology must be overcome for the translation of EVs into the clinic; in particular, in understanding their biogenesis and mechanism of action within the tumour microenvironment. The prolyl isomerase Cyclophilin A is overexpressed in a large variety of cancers and is associated with an aggressive phenotype of metastasis and chemoresistance. Unpublished data from our lab revealed that loss of CypA expression significantly reduced tumour growth and metastasis in vivo supporting a role in tumour progression. In this study, potential functions of CypA in EV biology and function are investigated. Methods: EV Isolation: Differential Ultracentrifugation, Optiprep Density Gradient EV characterization: Nanosight Tracking Analysis, Flow cytometry, Transmission Electron Microscopy, Mass spectrometry EV identification: Western Blot, Co-immunoprecipitation Results: CypA is found to be enriched in cancer-derived EVs in a range of solid and haematopoietic malignancies. In addition, CypA is predominantly found in EVs within a specific density range. Moreover, homozygous loss of CypA expression reduces the number of EVs within a specific size range. Investigation of CypA interacting proteins by mass spectrometry reveals potential functions in EV cargo loading. Summary/Conclusion: This study reveals a potential role for CypA in EV biogenesis, and highlights its potential as a novel EV target for the prevention of tumour progression. Significance of this study is that CypA could be a potential target for EV release. This work contributes to the understanding of CypA-dependent EV subtype for its biology and function during cancer metastasis and may reveal novel strategies for the generation of targeted EV subtype therapeutics. Funding: UCD-CSC Scholarship (not include travel funding). PT05.04=OWP2.12 Identification of a protein that presumably controls bacterial vesiculation in response to the extracellular environments Fumiaki Yokoyama ^a, Jun Kawamoto^a, Chen Chen^a, Tomoya Imai^b and Tatsuo Kurihara^a ^aInstitute for Chemical Research, Kyoto University, Uji, Japan; ^bResearch Institute for Sustainable Humanosphere, Kyoto University, Uji, Japan Introduction: Many bacteria utilize extracellular membrane vesicles (EMVs) for survival in their growing environments through communication with others, pathogenesis and biofilm formation. Therefore, the amounts and the components of EMVs should be tuned in response to the conditions. Although several vesiculation mechanisms are suggested, little is known how bacteria control vesiculation in response to the environments. A bacterium Shewanella sp. HM13 has 9-fold higher lipid-secretion capability in EMV fractions than Escherichia coli, and its EMVs contain a major protein (P49), which is not required for vesicle production. We used mutant EMVs that lack P49 to identify minor components of EMVs that may control vesiculation. Methods: EMVs were subjected to 2D gel-based proteomics by peptide mass fingerprinting. Within the identified proteins, the function of a sensor protein homolog, HM1275, was analysed by swarming assay and lipid-staining to quantify EMVs produced in various media. Changes in the number of EMVs depending on culture media were quantified by tunable resistive pulse sensing method. Results: A protein with a PAS domain and a methyl-accepting chemotaxis protein (MCP) sensing domain, HM1275, was identified in the EMVs. Although some MCPs are related to flagellar motility by binding some attractants, the flagellar motility of Delta-hm1275 was not significantly different from that of WT. Although the amounts of EMVs produced by WT were increased in response to the concentration of casamino acids in poor nutrient medium, those by Delta-hm1275 were not. Summary/conclusion: A putative sensor protein, HM1275, was identified in EMVs and may recognize the extracellular environments by binding signal molecules in casamino acids to control vesiculation. Although further studies are required to reveal the signals and the sensing pathways, the results obtained in this study indicate that bacterial vesiculation is controlled by extracellular environments, and artificial control of vesiculation with extracellular signals would be useful in applications such as suppression of vesicle-dependent pathogenicity. Funding: Japan Society for Promotion of Science Research Fellowship for Young Scientists PT05.05=OWP2.13 Prokaryotic BAR domain-like protein BdpA promotes outer membrane extensions Daniel A. Phillips ^a, Lori Zacharoff^b, Cheri Hampton^c, Grace Chong^b, Brian Eddie^d, Anthony Malanoski^d, Shuai Xu^b, Lauren Ann Metskas^e, Lina Bird^f, Grant Jensen^e, Lawrence Drummy^c, Moh El-Naggar^b and Sarah Glaven^d ^aAmerican Society for Engineering Education – U.S. Naval Research Laboratory, Washington, USA; ^bUniversity of Southern California, Los Angeles, USA; ^cMaterials and Manufacturing Directorate, Air Force Research Laboratory, Dayton, USA; ^dU.S. Naval Research Laboratory, Washington, USA; ^eCalifornia Institute of Technology, Pasadena, USA; ^fNational Research Council, Washington, USA Introduction: Bin/Amphiphysin/RVS (BAR) domains belong to a superfamily of membrane-associated coiled-coil proteins that influence membrane curvature. BAR domains are ubiquitous in eukaryotes and associated with membrane curvature formation, vesicle biogenesis/trafficking, protein scaffolding and intracellular signalling. While advances in protein domain prediction have facilitated the identification of several BAR domain proteins, they have yet to be characterized in bacteria. Here, we identified a putative BAR domain-containing protein enriched in the outer membrane vesicles (OMVs) of Shewanella oneidensis MR-1, a dissimilatory metal-reducing bacteria known to produce outer membrane extensions (OMEs) that are suspected to facilitate long distance extracellular electron transfer (EET) but whose physiological relevance and mechanism of formation remain unknown. Methods: Purified S. oneidensis OMVs were prepared by filtration and ultracentrifugation for comparative proteomics with cell-associated outer membrane proteins or for electrochemical measurements. Protein domains were predicted using HMMSCAN and CDD-search. OME formation and phenotype analyses were performed in situ by confocal and cryo-electron microscopy. Results: The putative BAR domain-like protein BdpA was highly enriched in OMVs compared to cell-associated outer membranes. During OME biogenesis, WT S. oneidensis OMEs progress from elongated vesicle chains to narrow, tubule-like extensions while ΔBdpA OMEs remain as disordered vesicle chains. Purified OMVs from these strains are electrochemically active, with redox signals consistent with multiheme outer membrane cytochromes, supporting the role of OMEs in EET. Heterologous BdpA expression promotes OME formation in Marinobacter atlanticus and Escherichia coli, suggesting BdpA membrane sculpting activity is inducible and transferrable. Summary/conclusion: The ability of BdpA to promote OME formation and maturation into tubules in vivo supports BdpA as a comparator for BAR domain protein activity in bacteria. Funding: US DoD Synthetic Biology for Military Environments (SBME) Applied Research for the Advancement of Science and Technology Priorities (ARAP) NSF Dimensions: DEB-1542527 US DOE: DE-FG02-13ER16415 PT06: EV Cancer Immunology Chairs: Jason Webber; Koichi FurukawaLocation: Level 3, Hall A15:30–16:30 PT06.01 Development of CD40L-modified small extracellular vesicles for the effective induction of anti-tumour immune response Wen Liu ^a, Yuki Takahashi^b, Masaki Morishita^c, Makiya Nishikawa^d and Yoshinobu Takakura^b ^aKyoto University, Kyoto, Japan; ^bGraduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; ^cKyoto Pharmaceutical University, kyoto, Japan; ^dTokyo University of Science, Noda, Japan Introduction: Tumour-derived small extracellular vesicles (sEVs) are anticipated to be a novel cancer vaccine because of their inherent encapsulation of tumour antigens. In this research, tumour-derived sEVs were modified with CD40 ligand (CD40L), which is a ligand for CD40 expressed on dendritic cells (DCs) and can activate DCs, in order to induce effective tumour antigen-specific immune response. Methods: B16-BL6 murine melanoma cells were selected as sEVs-producing cells. Plasmid vector encoding a fusion protein of CD40L and lactadherin (LA), named as CD40L-LA, was constructed. B16-BL6 cells were transfected with the CD40L-LA-expressing plasmid vector and CD40L-modified sEVs (CD40L-sEVs) were collected from the culture medium of the transfected cells. The collected sEVs were characterized by using western blot, zeta sizer and transmission electron microscope (TEM). CD40L-sEVs labelled with PKH67 were added to DCs and the uptake of CD40L-sEVs was evaluated by flow cytometer. CD40L-sEVs were added to DCs and the cytokine release from the cells was measured by ELISA. Presentation of melanoma antigens contained in sEVs were evaluated by measuring cytokine release from melanoma antigen gp100-specific T cells, which were co-incubated with CD40L-sEVs added DCs. The concentrations of cytokines in the culture medium were determined using ELISA. Results: The negatively charged sEVs with a diameter of approximately 100 nm were successfully modified with CD40L. CD40L-sEVs were more efficiently taken up by DCs than unmodified sEVs. DCs added with CD40L-sEVs produced more TNF-alpha and IL-12 than those added with unmodified sEVs. Moreover, CD40L-modification of sEVs improved the melanoma antigen presentation efficiency of DCs, which was demonstrated by increased IL-2 secretion from melanoma antigen gp100-specific T cells. Summary/Conclusion: It was shown that CD40L-modified sEVs can be used to induce effective anti-tumour immune response. PT06.02 Development of Interferon γ-loaded tumour cell-derived extracellular vesicles applicable to cancer vaccine therapy Naoki Nakagawa ^a, Yuki Takahashi^b and Yoshinobu Takakura^b ^aGraduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; ^bGraduate School of Pharmaceutical Sciences, Kyoto University, kyoto, Japan Introduction: Extracellular vesicles (EVs) contain various substances such as proteins and nucleic acids derived from their producing cells. As tumour cell-derived EV (TEV) contains tumour antigens, TEV is expected to be used as a cancer vaccine. However, since the immune activation ability of TEV is low, it is difficult to induce effective anti-tumour immunity by simple administration of TEV alone. Hence, in this study, we attempted to enhance the immune activation ability of TEV by loading Interferon (IFN)-γ. Methods: A plasmid vector encoding a fusion protein of lactadherin that specifically bind to phosphatidylserine contained in EV membrane and mouse IFN-γ was prepared and the vector was transfected into a mouse melanoma cell line B16BL6 cells. Then, IFN-γ-loaded TEV (γ-TEV) was collected from the supernatant of the transfected cells by ultracentrifugation. IFN-γ loaded on the collected TEVs was detected by Western blotting and ELISA. IFN-γ biological activity of IFN-γ loaded on γ-TEV was evaluated by a reporter assay. In addition, γ-TEV was added to the mouse dendritic cell line, DC 2.4, and mRNA and protein expression levels of antigen presentation-related genes were analysed using RT-qPCR and FACS analysis. Finally, splenocytes of mice that had received intradermal administration of γ-TEV were collected and the amount of IFN-γ produced from the splenocytes incubated with B16BL6 antigens was measured. Results: It was confirmed that IFN-γ was successfully loaded to TEV. In addition, the reporter assay confirmed that the biological activity of IFN-γ was retained in γ-TEV. Addition of γ-TEV to DC 2.4 increased mRNA and protein expression of MHC class I and CD86 compared to TEV alone group, which suggests that immune activation ability of TEV was increased by loading IFN-γ. Furthermore, in the splenocytes assay, the amount of IFN-γ production was significantly increased in the γ-TEV administration group compared with the group administered with simple mixture of IFN-γ and unmodified TEV. Summary/Conclusion: These results indicated that IFN-γ loading to TEV is an effective approach for cancer immunotherapy using TEV. PT06.03 Apoptotic neuroblastoma derived extracellular vesicles can prime mesenchymal stem cells to decrease regulatory T cells differentiation Anita KY. Li and Godfrey Chan The University of Hong Kong, Hong Kong, Hong Kong Introduction: There are many ongoing studies investigating tumour derived extracellular vesicles (EVs). Yet in cancer patients receiving chemotherapy, a majority of the tumour are undergoing apoptosis and the difference between health cancer and dying cancers EVs are still unknown. Apoptotic tumour cells can secrete EVs containing different messages to the tumour microenvironment and effect the surrounding cells in a different way. Mesenchymal stem cell (MSC) is a heterogeneous multipotent stem cell found within the tumour microenvironment and can regulating the immune system. The aim of this study is to investigate the role of apoptotic EVs on mesenchymal stem cell immunomodulatory function in a tumour microenvironment. Methods: EVs were obtained from both healthy SK-N-LP neuroblastoma cell line and those treated with the chemo drug cisplatin for 24 h. EVs were isolated from ultracentrifugation at 16,000 g for larger EVs and 100,000 g for smaller EVs. The characterization of the different populations of EVs was performed by western blot and nanoparticles tracking analysis. Neuroblastoma derived EVs were then co-cultured with immortalized human MSC (hTMSC) for 48 h. The immunomodulatory function of hTMSC was determined by their effect on T cells isolated from PBMC. Results: T cells co-cultured with hTMSC have an increase in FoxP3 expression whereas hTMSC that has been primed with apoptotic EVs from neuroblastoma showed a significant decrease in FoxP3 expression. The DAMP molecule HMGB1 was found to be present in apoptotic EVs, whilst being absent in healthy neuroblastoma EVs. Summary/Conclusion: Although MSCs are commonly known to have an immunosuppressive function, after the uptake of EVs derived from apoptotic neuroblastoma, MSC was able to switch to an immunostimulatory phenotype and decreasing Treg differentiation. Dying tumour cells may package danger signals and alarmins in their EVs thereby activating immune response in the tumour microenvironment. Funding: The Edward & Yolanda Wong Research Fund PT06.04 Chronic Lymphocytic Leukaemia-derived small extracellular vesicles: a potential strategy for immune escape Ernesto Gargiulo ^a, Sandrine Pierson^b, Bassam Janji^a, Jérôme Paggetti^a and Etienne Moussay^a ^aLuxembourg Institute of Health (LIH), Department of Oncology, Luxembourg, Luxembourg; ^bLuxembourg Institute of Health (LIH), Department of Oncology, Luxembourg, Luxembourg Introduction: Chronic Lymphocytic Leukaemia (CLL) is the most common adult leukaemia and characterized by the accumulation of abnormal B lymphocytes. CLL cell survival and proliferation are highly dependent on interactions with the microenvironment. Thus, to identify effective strategies to impair tumour proliferation, it is essential to understand the communication between CLL and surrounding tissues. Methods: To obtain a biological representation of small extracellular vesicles (small Evs) in the tumour microenvironment, we established a new protocol allowing us to isolate highly pure small Evs directly from the spleen of leukemic mice. Small Evs quality and sample purity were evaluated with qNano (TRPS principle), western blot and conventional bead-based flow cytometry. Next, we screened a wide range of immune checkpoint ligands on the surface of CLL-derived small Evs and corresponding receptors on the surface of T cells. Results: We have succeeded in isolating small Evs generated by CLL cells in vivo. Our screen suggested the presence on immune checkpoint ligands directly anchored on tumour-derived small Evs. Furthermore, we identified a promising pair ligand-receptor potentially implicated in immune escape. Validation of candidates from the screen is currently being performed through FACS, iFACS and EM. These techniques will allow us to better define tumour-derived small Evs populations presenting different immune checkpoints and to visualize single small Evs with high resolution. Summary/Conclusion: In this project, we aimed to isolate and characterize CLL-derived small Evs to define their involvement in tumour development, with focus on the evaluation of their impact on CLL immune escape. Altogether, this study will give insight into the specific immune and stromal cells involved in CLL development, with emphasis on their involvement in tumour-derived small Ev-mediated tumour immune escape. Funding: This project is funded by the Fonds National de la Recherche (FNR) INTER/DFG/16/11509946/EV-RNA/Moussay. Sandrine Pierson and Jérôme Paggetti are supported by the FNR INTER/DFG/16/11509946/EV-RNA/Moussay. Ernesto Gargiulo is supported by the grant FNR Luxembourg PRIDE15/10675146/CANBIO. PT06.05 Interaction via exosome miRNAs between myelodysplatic cell and normal Treg Tatsuki Shibuta, Yukichi Takada and Tsukuru Umemura International University of Health and Welfare, Okawa City, Japan Introduction: Myelodysplastic Syndrome (MDS) is a clonalhematopoietic disease and develops leukaemia in some cases. Thus, MDS is a malignant hematopoietic disease and its prevalence ratio is increasing in Japan. Hematopoietic microenvironment such as bone marrow niche is a crucial factor for maintaining leukaemic stem cells. To understand mechanisms of interactions between leukaemic stem cells and microenvironment is important for the treatment of hematopoietic malignancies. In this study, to develop the new therapies and diagnostic methods for MDS, we focused on the effect of exosomes released from MDS cells on peripheral T lymphocytes. Methods: MDS cell line (MDS-L) was kindly provided by Kasawaki Medical University and normal peripheral blood mononuclear cells were obtained from healthy volunteer donors. Exosomes from MDS cells were purified by using miRCURY Exosome Cell/Urine/CSF Kit and labelled by PKH67. Extracted miRNAs were analysed by microarray method (Genopal, Mitsubishi Chemical, Japan). Cell surface antigens were analysed by FACS Aria II and fluorescence conjugated antibodies. Results: miRNA-microarray analysis showed that nine miRNAs were abundant in exosomes from MDS cells and were not detected in MDS cells. Exosomes labelled with PKH67 dye were added to liquid culture of regulatory T cells (Treg) that were sorted from normal peripheral blood. The exosomes were detected in cytosol of Treg by fluorescent microscopy. Microarray analysis of miRNAs in Treg intaking MDS-exosomes showed that significant increases of 9 miRNAs in MDS-exosomes. The conditioned medium of MDS-exosomes treated Treg culture reduced the population of activated CD4 cells (CD38 positive cells was 39%; control 68%). Summary/Conclusion: Our data suggested that exosomes from MDS cells affected the function of regulatory T cells via miRNA transfer. MDS exosomes may effect on immune cells to avoid the exclusion from cancer-immune system, and may be a target for the new therapies or diagnostic methods. Funding: This work was supported in part by a grant from the Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number: JP17K09020 and 17H07059). PT06.06 Mechanism of antitumor immunity activation by ‘artificial neoantigen’-presenting exosomes Yoshiyuki Koyama ^a, Tomoko Ito^a, Masazumi Eriguchi^a, Aya Hasegawa^b, Wakana Ouchi^c, Toshio Inaba^b and Kikuya Sugiura^b ^aJapan Anti-tuberculosis Association, Shin-Yamanote Hospital, Tokyo, Japan; ^bOsaka Prefecture University, Osaka, Japan; ^cOsaka Prefecture University, Tokyo, Japan Introduction: Tumour-derived exosomes are known to have same antigens as the parent tumour cells, and were expected as cancer vaccines. However, treatment with those exosomes often failed to elicit antitumor immune responses, probably owing to a weak immunogenicity of the tumour-associated antigens (TAAs). TAAs can be divided into two categories, overexpressed self-antigens and tumour-specific mutated neoantigens. Recently, it was found that immunotherapy was effective only in patients whose tumours had neoantigens with high immunogenicity. However, most tumours do not have such efficient neoantigens. To overcome the disadvantage, we have developed an “artificial neoantigen strategy“. Previously, we prepared the exosomes expressing strong bacterial antigen, as an “artificial neoantigen” by transformation of the original cultured cells with a gene of the Mycobacterium tuberculosis antigen, early secretory antigenic target-6 (ESAT-6). Injection of the exosomes induced significant antitumor activity in tumour bearing mice. Here we investigated the mechanism of antitumor immunity activation by the ”artificial neoantigen”-presenting exosomes. Methods: Mouse B16 melanoma cells were transfected with ESAT-6, and secreted ESAT-6 antigen-presenting exosomes (ESAT-Ex) were isolated. Cultured mouse DCs were treated with the exosomes and expression of costimulatory molecules and cytokines was assessed. Antitumor activity of the ESAT-Ex-stimulated DCs was evaluated in tumour bearing mice. In vivo immune activating ability of ESAT-Ex was investigated by co-incubation of the lymphocytes taken from the ESAT-Ex-treated mice with B16 tumour cells. Immuno-stimulation by the fibroblasts-secreted ESAT-Ex was also studied. Results: The DCs stimulated with ESAT-Ex showed enhanced CD80 and CD86 presentation, and exhibited significantly improved antitumor activity in tumour-bearing mice. When the lymphocytes harvested from the mice injected with ESAT-Ex were co-incubated with B16 cells, they intensely accumulated around the tumour cells, and secreted higher level of IFN-gamma. Increased uptake of thymidine was also observed. Summary/Conclusion: “Artificial neoantigen”-presenting exosomes effectively stimulated DCs and evoked antitumor immunity. They are expected as novel cancer vaccines. Funding: This work was supported by JSPS KAKENHI Grant Number 16K01394. PT06.07 Outer membrane vesicles of Tannerellaforsythia induce inflammatory response in periodontal tissue cells Sunjin An Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University, Seoul, Republic of Korea Introduction: Tannerella forsythia, a Gram-negative oral bacterium, is one of the major periodontal pathogens which can cause inflammatory responses. Inflammasome is crucial for host defence against pathogens, but excessive inflammasome activation can lead to tissue damage. Outer membrane vesicles (OMVs) are derived from the cell envelope of Gram-negative bacteria. OMVs can contain DNA, RNA, lipopolysaccharide, proteins, toxins and peptidoglycan. T. forsythia induces maturation of IL-1α/IL-1β and cell death via activation of caspase-1/4 in THP-1 macrophages and human gingival fibroblasts (HGFs). The aim of this study was to investigate whether T. forsythia OMVs are involved in inflammasome activation which may contribute to periodontitis, a chronic inflammatory disease. Methods: T. forsythia OMVs were isolated using Exo-bacteria OMVs isolation kit. THP-1 macrophages differentiated with PMA and HGFs were treated with T. forsythia and OMVs at various does. The expression of caspase-1/4 and pro-inflammatory cytokines was determined by immunoblotting and ELISA, respectively. Cell death was measured by LDH cytotoxicity assay kit. Results: T. forsythia OMVs activated caspase-1 and −4, resulting in increased IL-1α and IL-1β release and inflammatory cell death. The OMVs also induced the expression of IL-6 and IL-8. Summary/Conclusion: The results indicated that T. forsythia OMVs may play an important role in inflammatory response in T. forsythia-infected cells. Funding: This work was supported by National Research Foundation of Korea grants (No. NRF-2018R1A5A2024418 and NRF-2018R1A2A2A05018558). PT06.09 Specific decrease of CD19+extracellular vesicles enhances post-chemotherapeutic CD8+T cell responses Fanghui Zhang, Xuefeng Fei and Zhijian Cai Institute of Immunology, School of Medicine, Zhejiang University, Hangzhou, China (People’s Republic) Introduction: Chemotherapy has long been related with induction of systemic immunosuppression. Systemic immunosuppression greatly affects chemotherapeutic antitumor effect. Therefore, amelioration of systemic immunosuppression following chemotherapy is necessary to improve post-chemotherapeutic antitumour immunity. Methods: CD19+EVs were isolated and identified by EM, NTA, FACS and western blotting. CD19+EVs functions on degrading ATP and their immunosuppressive functions were assessed in vitro and in vivo. The effects of CD19+EVs on antitumour effect of chemotherapy were detected by transfer of exogenous EVs into tumour mice or in Rab27a or Hif1a conditional knockout tumour mice. The effects of CD19+EVs on antitumor effect of chemotherapy were also evaluated in humanized NSG mice by knocking down Rab27a with inactivated EBVs loading with Rab27a siRNA. Results: CD19+extracellular vesicles (EVs) from B cells hydrolyse ATP from chemotherapy-treated tumour cells into adenosine by CD39 and CD73, resulting in impaired CD8+T cell responses. Serum CD19+EVs increase in tumour mice and patients. Patients with fewer serum CD19+EVs have a better prognosis following chemotherapy. Up-regulated HIF-1α promotes B cells releasing CD19+EVs by inducing Rab27a mRNA transcription. Rab27a or HIF-1α deficiency in B cells inhibits CD19+EV production and strikingly improves chemotherapeutic antitumor effect. Knockdown of Rab27a in B cells by inactivated Epstein-Barr viruses carrying Rab27a siRNA greatly improves chemotherapeutic efficacy in humanized NSG mice. Summary/Conclusion: Our findings unravel a mechanism underlying systematic immunosuppression after chemotherapy. Combination of chemotherapy and iEBVs/Rab27a siRNA holds high potential for cancer treatment. Funding: This work was supported by grants from the National Key Basic Research Program of China (2015CB943301), the National Key R&D Program of China (2016YFA0501800), and the National Natural Science Foundation of China (31770951 and 31670877). PT07: EVs in Acute and Chronic Inflammatory Disorders Chairs: Eric Boilard; Aleksandra GaseckaLocation: Level 3, Hall A15:30–16:30 PT07.01 Circulating microvesicles as potential biomarkers of Acute Respiratory Distress Syndrome in Sepsis Marcelo Fernando do Nascimento^a, Ana Cláudia Aparecida Santos Nussbaum^b, Ludhmila Abrahao Hajjar^c, Luciano Cesar Pontes Azevedo^d, José Mauro Vieira Junior^d, Daniel Deheinzelin^d, Roger Chammas^c, Juliana Monte. Real ^c ^aInstituto do Servidor Publico Estadual, Santo André, Brazil; ^bHospital do Coracao, sao paulo, Brazil; ^cInstituto do Cancer do Estado de Sao Paulo, Sao Paulo, Brazil; ^dHospital Sirio-Libanes, Sao Paulo, Brazil Introduction: Acute respiratory distress syndrome (ARDS) is a clinical condition of sudden respiratory failure in critically ill patients. ARDS-related mortality rate is higher when is associated with Sepsis (>50%). Recently, we screened 754 miRNAs and discovered a different cargo transported by circulating extracellular vesicles (EVs) and exosomes from patients with sepsis, remarkably in those who progressed to death. The early sequence of events of respiratory failure after the onset of sepsis are still unknown. Our hypothesis is that lung should signal through EVs that it is being affected by SIR. Methods: Blood samples were obtained from septic patients with (n = 8) and without ARDS (n = 5) at 24 h of intensive care unit (ICU) admission and 3 days later at Sirio-Libanes Hospital. Pulmonary originated sepsis was not considered. Eight patients under mechanical ventilation (MV) without pulmonary disease and 12 healthy volunteers were used as controls. Plasma was 0.22 µM filtered, EVs were isolated by ultracentrifugation and analysed by nanoparticle tracking analysis. Based on our previous data, 48 miRNAs were measured by Taqman Low Density PCR array and normalized by RNU6. Results: The main population of EVs peaked at size of 155–165 nm with no difference in the mean concentration between groups. Patients with sepsis + ARDS showed a significant decrease in plasma EVs 3 days after ICU stay (234 to 137 x 10e8/mL, p = 0.0175). Compared to healthy donors, sepsis promotes an even significant alteration of EVs-miRNAs when it is associated with ARDS. Comparing all samples from patients with sepsis + ARDS to sepsis only, nine miRNAs are transported in smaller amounts: miR-766 (−35.7, p = 0.002), miR-127 (−23.8, p = 0.001), miR-340 (−13.5, p = 0.006), miR-29b (−12.8, p = 0.001), miR-744 (−7.1, p = 0.05), miR-618 (−4.0, p = 0.02), miR-598 (−3.8, p = 0.035), miR-1260 (−2.5, p = 0.035); and miR-885-5p is expressed at higher levels (9.5; p = 0.028). In paired samples, the set of altered miRNAs is generally different (p < 0.05) between sepsis + ARDS (miR-148a, −193a-5p, 199a-3p, −222, −25, −340, 744) or sepsis only (miR-1183, −1267, −1290, −17, −192, −199a-3p, −25, −485-3p, −518d, −720). Summary/Conclusion: Circulating EV-miRNAs cargo could be potential biomarkers of lung inflammation during sepsis in patients who will require MV. Funding: FAPESP. PT07.02 Innate/ inflammatory cross talk between macrophages (Mps) and RPE cells are mediated by exosomes secreted by RPE cells: Proposal of new trait for the pathogenesis of age-related macular degeneration (AMD) Atsushi Mukai ^a, Eiko Ito^a, Morio Ueno^a, Shigeru Kinoshita, Chie Sotozono^a and Junji Hamuro^a ^aDepartment of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; ^bDepartment of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan Introduction: The pathogenesis of AMD is aggravated by chronic inflammation. Intact RPE down-regulates the production of TNF-alpha by choroid-infiltrating Mps, whereas degenerated RPE by oxidative stress were devoid of this regulatory function. Subsequently, locally produced TNF-alpha induces the production of some pro-inflammatory cytokines and angiogenic factor VEGF by RPE (Yamawaki et al., 2016). This implies that innate/inflammatory cross talk between Mps and RPE may be the indispensable trait for AMD pathogenesis. The purpose of this study is to elucidate the signal that causes up-regulated TNF-alpha production in congenital / inflammatory crosstalk between Mps and RPE. Methods: Mps cell line RAW 264.7(RAW) was co-cultured with primary RPE taken from C57BL/6 mice. Some cytokines in the culture supernatants (CSs) were quantified by ELISA. The expression profiles of complement-associated genes, TNF-alpha, and angiogenesis-associated genes (VEGF & PEDF) were analysed by qRT-PCR. For the preparation of exosomes (Exo), CSs were harvested after co-cultures of RAW with primary RPE, then Exo in each CSs were purified by either EVsecondTM or ultracentrifugation. The incorporation of the Exo either into RPE or RAW was histologically quantified using Qdot 655 streptavidin conjugated biotinylated Exo. Results: Elevated levels of CD63 positive Exo in co-cultures were detected by western blot or FACS analysis. The produced Exo in co-culture CSs were incorporated solely into RAW, but not into RPE. The semi-purified Exo, but not the Exo depleted residual CSs enhanced the secretion of MCP-1 and IL-6 in co-culture of Mps and RPE, while the enhancement of VEGF are similarly detected by the Exo deprived residual CSs. Most remarkable elevation was observed in TNF-alpha production by RAW in a dose-dependent manner even in the absence of RPE. The down-regulated TNF-production by RAW in the presence of RPE was not reconstituted by the addition of Exo even in the co-culture. Summary/Conclusion: Exosome displays a critical role in the triggering of vicious inflammatory cytokines cycle through the elevation of TNF- production by Mps. Currently, in order to construct an experimental system closer to the pathology of AMD, we are studying a co-culture system using human Mps and human iPS-derived RPE. PT07.03 Epithelial exosomes regulated by phosphatase Shp2 promote macrophage activation Yuefei Zhang ^a, Yiqing Li^b, Dacheng Gong^b, Hongqiang Cheng^b, Xue Zhang^c and Yuehai Ke^b ^aZhejiang University, Hangzhou, China (People’s Republic); ^bZhejiang University, School of Medicine, Hangzhou, China (People’s Republic); ^cZhejiang University, School of Medicine, Hangzhou, China (People’s Republic) Introduction: Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are life-threatening diseases that are associated with high mortality rates due to treatment limitations. Increasing researches suggest exosomes play an important role in pathogenesis, diagnosis and treatment of ALI. However, it’s not clear how exosomes are formed, secreted, transferred during ALI. Phosphorylation of signalling proteins are reported to control exosome biogenesis (e.g. syntenin phosphorylation promotes exosome formation). Shp2 is a widely expressed cytoplasmic phosphatase which can regulate signalling pathway by its dephosphorylation function. Here we reveal that Shp2 inhibits the biogenesis of epithelial exosomes which have proinflammatory effects on macrophages during ALI. It’s uncovered in our study that Shp2 is a protective factor of ALI by inhibiting release of proinflammatory epithelial exosomes. Methods: Exosomes were isolated by differential ultracentrifugation and filtration, and they were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blot (e.g. CD9, CD63, CD81, ALIX, TSG101). In vitro transwell system for exosome transfer model indicated the direction of exosome transfer. Nanoscale flow cytometry (CytoFLEX) was used for detecting exosome subpopulation. Results: Exosomes were increased in Bronchoalveolar Lavage Fluid (BALF) of LPS-induced ALI murine model. In vitro transwell system revealed that exosomes were transferred from epithelial cells to macrophages in inflammation environment. Shp2 was revealed to inhibit the biogenesis of epithelial exosomes without changing their size and subpopulation. Adaptor protein Gab2, which can bind Shp2, was found to interact with Syntenin. It suggests that with the help of adaptor Gab2, Shp2 was involved in dephosphorylating syntenin whose phosphorylation can facilitate exosome biogenesis. Shp2-disruption derived epithelial exosomes promoted macrophage inflammation, thus aggravating ALI. Summary/Conclusion: Our study shows that phosphatase Shp2 inhibits proinflammatory epithelial exosome release, which can promote M1-macrophage polarization. It offers a potential target for ALI mechanism study and treatment. PT07.04 Detection of CD11b-expressing exosomes in plasma of mice with sepsis Yasunori Fujita, Kyojiro Kawakami and Masafumi Ito Research Team for Mechanism of Ageing, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Japan Introduction: Cells communicate with each other through extracellular vesicles including exosomes, which contain host cell-derived molecules such as proteins, lipids and nucleic acids. Secreted exosomes migrate not only to neighbouring cells but also to distant organs. Monocyte and macrophage have been reported to secret exosomes that modulate immune responses. However, the characteristics of monocyte/macrophage-derived exosomes in blood during systemic immune response remain largely unknown. In this study, we characterized exosomes released from monocyte/macrophage-like cells and determined the temporal change in monocyte/macrophage-derived exosomes in plasma of mice with sepsis. Methods: Exosomes collected by ultracentrifugation from the conditioned medium of lipopolysaccharide (LPS)-stimulated murine monocyte/macrophage-like RAW264.7 cells were subjected to quantitative proteomic analysis using iTRAQ labelling and LC-MALDI-TOF/TOF. Plasma exosomes isolated from LPS-injected mice were analysed by Western blot analysis. CD11b-expressing exosomes in plasma were measured by sandwich ELISA. Plasma TNF-α level was determined by ELISA. Results: Proteomic analysis showed that monocyte/macrophage marker proteins such as CD11b, CD14 and F4/80 were detected in exosomes from RAW264.7 cells. Glucose metabolism-related proteins including GLUT1, PKM2 and GAPDH increased in exosomes from LPS-stimulated cells compared with those from non-treated cells. Western blot analysis demonstrated that GLUT1 and CD11b were significantly increased in plasma exosomes from LPS-injected mice. After LPS stimulation, TNF-α transiently increased, whereas CD11b-expressing exosomes increased and remained high in plasma of mice with sepsis. Summary/Conclusion: We characterized monocyte/macrophage-derived exosomes in plasma of mice with sepsis and developed a sandwich ELISA for detection of CD11b-expressing exosomes in plasma, which could be a novel marker for systemic immune response as well as sepsis. Funding: JSPS KAKENHI Grant Number JP17K01888. PT07.05 Systemic inflammatory activity and proteome analysis of extracellular vesicles from faeces Kyongsu Park^a, Jaewook Lee^b, Yein Jun ^a, Daekyum Kim^a, Jungwook Kim^a and Yong Song Gho^c ^aPohang University of Science and Technology (POSTECH), Pohang, Republic of Korea; ^bDepartment of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea; ^cDepartment of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea Introduction: Substantial quantities of bacteria reside in the gastrointestinal tract. Severe inflammatory responses are induced when the bacteria went through the peritoneum from the gastrointestinal tract. In this study, extracellular vesicles isolated from faeces (fEVs) were assessed to see whether they could mediate inflammatory responses. In addition, proteomic compositions of fEVs were further investigated. Methods: The faeces of wild-type mice were utilized to isolate fEVs. The fEVs were characterized with transmission electron microscopy, dynamic light scattering, ELISA, and Western blot. The fEVs were intraperitoneally administered into the mice, and the number of infiltrated cells as well as the concentrations of TNF-α and IL-6 were measured from the peritoneal lavage fluid, serum, and bronchoalveolar lavage fluids. Proteomic analyses on the fEVs were conducted by the combination of one-dimensional SDS-PAGE and LC-MS/MS. Results: Significant amounts of fEVs were isolated from mouse faeces, and the fEVs were derived from bacteria and host cells. Upon intraperitoneal administration, the fEVs mediated peritoneal, systemic, and pulmonary inflammation by increasing the numbers of infiltrated immune cells and the pro-inflammatory cytokines such as TNF-α and IL-6 in the peritoneal lavage fluid, serum, and bronchoalveolar lavage fluid. Proteomic analyses on the fEVs identified a total of 295 proteins, comprising 222 bacterial proteins and 73 murine proteins. Summary/Conclusion: The fEVs derived from bacterial and host cells could mediate local and systemic inflammation, and composed of bacterial and host proteins. These results shed lights on the roles of commensal bacterial EVs in the pathogenesis of inflammatory diseases. Funding: National Research Foundation of Korea (NRF) Herman Krefting Foundation for Allergy and Asthma Research, Lundberg Foundation PT07.06 Opioid-mediated release of astrocytic EV miR-23 induces pericyte migration and blood-brain barrier breach Shilpa Buch, Ke Liao, Fang Niu and Guoku Hu University of Nebraska Medical Center, Omaha, USA Introduction: Pericytes are important constituents of the cerebrovascular unit and play a key role in maintaining the integrity of the blood-brain barrier. It is well recognized that drugs of abuse such as opioids can result in breach of the BBB, ultimately leading to enhanced monocyte transmigration and ensuing neuroinflammation. Mechanism(s) by which pericytes contribute to morphine-mediated neuroinflammation, however, remains less understood. Methods: EVs were isolated from morphine-stimulated mouse/human primary astrocytes using the standard differential ultracentrifugation method and characterized by transmission electron microscopy, NanoSight & western blot analyses. Among the various miRs dysregulated in morphine-stimulated astrocyte EV cargo, miR-23 was found to be upregulated by real-time PCR. Confocal microscopy identified uptake of astrocytic EVs by pericytes. Functional assessment of astrocytic EV uptake by pericytes involved cell migration using Boyden chamber and wound healing assays. Additionally, an in vitro 3D model comprising of pericytes and human endothelial cells was also used to assess astrocyte EV-mediated migration of pericytes in presence of morphine. Results: Exposure of astrocytes to morphine induced the expression and secretion of miR-23 in the EVs, which, upon uptake by the pericytes resulted in their migration. Additionally, in the pericytes that had taken up morphine stimulated astrocyte EVs, there was downregulation of phosphatase and tensin homologue (PTEN), a target of miR-23. Summary/Conclusion: Our findings indicate that morphine-mediated dysregulation of miRNA expression in the CNS involves astrocyte-pericyte communication via the extracellular vesicles, leading, in turn, to loss of pericyte coverage at the BBB. Funding: This work was supported by grants DA040397, [23]MH112848 (S.B.) and [24]DA042704, [25]DA046831 (G.H.) from the National Institutes of Health. The support by Nebraska Center for Substance Abuse Research is acknowledged. PT07.07=OWP2.15 Diagnostic microRNA biomarkers from circulating extracellular vesicles for early detection of pneumonia and severe secondary complications Stefanie Hermann ^a, Benedikt Kirchner^b, Dominik Buschmann^c, Melanie Märte^d, Florian Brandes^d, Stefan Kotschote^e, Michael Bonin^f, Marlene Reithmair^g, Matthias Klein^h, Gustav Schelling^d and Michael Pfaffl^d ^aDivision of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany; ^bAnimal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Freising, Germany; ^cTUM School of Life Sciences Weihenstephan, Division of Animal Physiology and Immunology, Freising, Freising, Germany; ^dDepartment of Anesthesiology, University Hospital, Ludwig-Maximilians-University Munich, München, Germany; ^eIMGM Laboratories GmbH, Planegg, Martinsried, Germany; ^fIMGM Laboratories GmbH, Planegg, Germany, Martinsried, USA; ^gInstitute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, München, Germany; ^hDepartment of Neurology, University Hospital, Ludwig-Maximilians-University Munich, München, Germany Introduction: Pneumonia remains one of the most deadly communicable diseases, causing three million deaths worldwide in 2016. Extracellular vesicles (EVs) are pivotal during signal transfer in the pathogenesis of inflammatory lung diseases. Since identifying pneumonia is particularly challenging in high risk groups (e.g. the elderly or infants), which often present with atypical symptoms and are at high risk for secondary complications such as sepsis or acute respiratory distress syndrome (ARDS), new approaches for early diagnosis are required. In this study we identified EV microRNAs (miRNAs) as potential biomarkers for inflammatory changes of the pulmonary tissue. Methods: Our study included 13 patients with community-acquired pneumonia, 14 ARDS patients, 22 patients with sepsis and 31 healthy controls. After precipitating EVs from 1 ml serum, total RNA was extracted. Subsequent to library preparation and small RNA-Seq, differential gene expression analysis was performed using DESeq2. Data were filtered by mean miRNA expression of ≥ 50 reads, minimum twofold up or down regulation and adjusted p-value ≤ 0.05. Results: The mean relative miRNA frequency varied slightly between the different groups and was highest in volunteers. Short sequences (< 16 nucleotides), probably degradation products from longer coding and non-coding RNA species, were predominantly detected in patients. Based on unsupervised clustering, patients could be distinctly separated from healthy individuals. Although 21 miRNAs were significantly regulated in all patient groups compared to healthy controls, different disorders showed unique miRNA expression profiles. Distinct miRNA subsets were identified, which are applicable to indicate disease progression from limited inflammation present in pneumonia to severe inflammatory changes as seen in ARDS and sepsis. Summary/conclusion: This study shows that EV miRNA biomarkers have potential for diagnosis of pneumonia and to indicate disease progression towards severe lung injury. Our findings are of clinical relevance, as the timely diagnosis of pneumonia can be challenging, and secondary complications such as ARDS and sepsis might be prevented by early intervention and treatment. Funding: This study was supported by the German Federal Ministry for Economic Affairs and Energy under the program “Zentrales Innovationsprogramm Mittelstand”. PT08: EVs in Metabolism and Metabolic Diseases Chairs: Sophie Rome; Alena IvanovaLocation: Level 3, Hall A15:30–16:30 PT08.01 Elevated levels of platelet and endothelial extracellular vesicles in type 1 diabetes, a cohort study of 236 patients Karin Bergen ^a, Katherina Aguilera Gatica^b, Fariborz Mobarrez^c, Gun Jörneskog^d, Håkan Wallén^b and Sara Tehrani^d ^aKarolinska Institutet, Department of Clinical Sciences, Danderyd University Hospital, Division of Nephrology, Stockholm, Sweden; ^bKarolinska Institutet, Department of Clinical Sciences, Danderyd University Hospital, Division of Cardiology, Stockholm, Sweden; ^cKarolinska Institutet, Department of Medicine, Rheumatology Unit, Karolinska University Hospital, Sweden, Stockholm; ^dKarolinska Institutet, Department of Clinical Sciences, Danderyd University Hospital, Division of Internal Medicine, Stockholm, Sweden, Stockholm, Sweden Introduction: We have recently presented data on increased levels of circulating extracellular vesicles (EVs) in patients with type 1 diabetes. In the same cohort, we have now analysed subpopulations of platelet- and endothelial EVs in relation to diabetic microangiopathy and sex. Methods: Two hundred and thirty-six patients (107 women) and 100 healthy controls matched for age, sex and body mass index (BMI) gave written informed consent to the study. Plasma platelet EV (PEV) and endothelial EV (EEV) levels were assessed using flow cytometry with labelled antibodies directed against platelet (CD42a and CD61) and endothelial specific (CD144 and CD62E) antigens. EV expression of procoagulant phosphatidylserine (PS) and tissue factor (TF) were assessed using lactadherin (lac) and CD142 antibody. The study was approved by the local ethics committee. Results: PEV and EEV levels with or without expression of procoagulant PS and TF were statistically higher among patients than in controls (p < 0.05 for all). The patients had about 50% higher PEV levels and up to a 50-fold increase in EEV levels compared to controls. No statistically significant differences were found between PEV or EEV levels in patients with or without clinical microangiopathy. Healthy women had lower PS+, PS− and total PEV levels compared to healthy men (p < 0.05 for all), whereas no differences between sex were found in the patients. PEV and EEV levels in patients did not correlate with glycaemic control (HbA1c), BMI, blood pressure, blood lipids or diabetes duration. Summary/Conclusion: Elevated levels of PEVs and EEVs found in patients with type 1 diabetes are unrelated to clinical microangiopathy, indicating that type 1 diabetes is a procoagulant state per se. Comparison of PEV levels between the sexes showed a more favourable phenotype in healthy women compared with healthy men, while no sex differences were found among patients. This could be linked to the loss of female protection against cardiovascular disease in type 1 diabetes. Funding: Berth von Kantzow Foundation, Swedish Diabetes Foundation, Wallenius Foundation, Swedish Heart-Lung Foundation, Foundation of Women and Health PT08.02 Role of extracellular vesicles in the regulation of inflammation and metabolism in obesity Takahisa Nakamura ^a, Ahlee Kim^b, Esam Salem^b, Kazutoshi Murakami^b and Vishnupriya Borra^b ^aCincinnati Children’s Hospiltal Medical Center, Cincinnati, USA; ^bCincinnati Children’s Hospital Medical Center, Cincinnati, USA Introduction: The worldwide prevalence of obesity has reached pandemic proportions. Obesity has strong inflammatory underpinnings, which are associated with the development of type 2 diabetes (T2D) and non-alcoholic steatohepatitis (NASH). However, the mechanisms by which obesity provokes aberrant inflammation have yet to be clearly defined. Extracellular vesicles (EVs), including exosomes and microvesicles, are a novel mode of tissue-to-tissue communication. Recent studies indicate that EVs are involved in many pathophysiological events including inflammatory responses and metabolic dysfunctions. We hypothesize that EVs play critical roles in the induction of obesity-associated aberrant inflammation and the development of metabolic diseases. Methods: To investigate the role of EVs in the pathogenesis of obesity, we have taken systematical approaches including novel computational methods, analyses of EVs collected from human obese patients undergoing bariatric surgery, utilization of novel mouse models monitoring cell type-specific EVs, and cellular-based EV functional assays. Results: Using novel computational methods, we have identified strong associations with EV-related genes in metabolic syndrome associated with T2D. Our analyses of EVs from adolescent obese patients undergoing bariatric surgery have shown that serum EV concentration is inversely correlated to metabolic improvements in glucose metabolism and inflammation post-surgery, with unique EVs’ extracellular RNA (exRNA) profiles. Further, our newly established mouse models monitoring specific cell type-derived EVs in vivo indicates that in obesity, EVs from metabolic tissues behave like a pathogen and induce inflammation. Summary/Conclusion: While the research of EVs has attracted much attention, therapeutic targeting and significance of EVs in metabolic diseases are still a controversial area of research. By utilizing our novel mouse models coupled with access to human samples, our systematical approaches allow to propose novel mechanisms by which pathologic EVs induce aberrant inflammation and deteriorate metabolism in obesity. PT08.03 Characterization of exosomal proteins derived from contracting skeletal muscle as potential mediators of beneficial metabolic effects of exercise Henrike Sell, Elisabetta De Filippo, Sonja Hartwig, Lucia Mastrototaro, Maria Apostolopoulou, Dominik Pesta, Julia Szendrödi, Hadi Al-Hasani, Stefan Lehr and Michael Roden German Diabetes Center, Düsseldorf, Germany Introduction: Exercise training improves glucose metabolism and insulin sensitivity supporting the concept that lifestyle modification is useful for patients to prevent and treat type 2 diabetes. Skeletal muscle also releases circulating factors following exercise affecting metabolism of other organs probably involving the release of exosomes. However, little is known about muscle-specific release of exosomes and the differential protein content of exosomes during exercise. Thus, we aimed to identify exosomal proteins regulated by exercise in vitro and in vivo and to discover pathways regulated by exosomal cargo. Methods: Exosomes from human skeletal muscle cells (hSkMC), contracted by electrical pulse stimulation (EPS), were isolated by differential ultracentrifugation/ultrafiltration. Exosomes were also isolated by size exclusion chromatography from plasma of patients with and without type 2 diabetes on high intensity interval training (HIIT). In order to allow reliable label-free quantification of the very limited muscle exosomal material, we performed proteomic profiling using data independent acquisition (DIA) on an OrbitrapTM Fusion Lumos instrument. Spectronaut TM Pulsar software was used to integrate spectral libraries and perform quantitative proteomic profiling of exosomes derived from different human primary cells as well as human serum and plasma. Results: EPS stimulated the release of exosomes from hSkMC and regulated the release of 408 exosomal proteins. Ingenuity pathway analysis (IPA) revealed significant regulation of, e.g. integrin, vascular endothelial growth factor, Liver X receptor/Retinoid X receptor and PI 3-kinase/Akt signalling by these proteins. HIIT regulated the amount of 62 exosomal plasma proteins in vivo, of which nine candidates were also similarly regulated by EPS in vitro. IPA links these exosomal proteins to pathways involved in metabolic diseases and lipid metabolism. Summary/Conclusion: We identified various exercise-regulated exosomal proteins with predicted metabolic effects. Further analysis of these novel tissue-specific candidates will help to better understand systemic metabolic effects of exercise. PT08.04 Transcriptome and proteome of extracellular vesicles derived from cellular targets of diabetic kidney disease Karina A. Barreiro ^a, Abigail Lay^b, Wei Liang^c, Xiaomeng Xu^d, Sihui Luo^d, Tillmann Bork^c, Richard Coward^e, Denis Delic^f, Tobias B. Huber^g and Harry Holthöfer^h ^aFIMM, University of Helsinki, Helsinki, Finland; ^bUniversity of Bristol, Bristol, UK; ^cUniversity Hospital Freiburg, Freiburg, Germany; ^dInstitute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland; ^eBristol Univ Medical School, Bristol, UK, Bristol, UK; ^fBoehringer Ingelheim Pharma GmbH & Co. KG, Biberach a.d. Riss, Germany; ^gIII. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, Hamburg, Germany; ^hProfessor Introduction: Kidney disease (DKD) is common, costly and the most feared complication of long standing diabetes. Its root causes remain unknown. Interestingly the characteristic changes in circulating glucose levels in this disease appear to alter the signature of extracellular vesicles as found in the urine. Here we wanted to explore the EV secretion pattern by key DKD target cells in the glomerulus (podocytes, endothelial and mesangial cells) and proximal tubular cells by harvesting the entire EV repertoire using Hydrostatic Filtration Dialysis (HFD). Methods: We used cell culture media from podocytes, proximal tubule, mesangial and glomerular endothelial cells in four conditions: (1) Insulin sensitive, (2) Insulin resistant, (3) Insulin receptor transfected and insulin sensitive, (4) Insulin receptor transfected and insulin resistant. EVs were isolated from 50 mL of cell culture media, respectively, by HFD. Quality of the EV yield was verified with negative staining Electron Microscopy (EM) and Western blotting (WB). Vesicle concentration was determined by Nanoparticle Tracking Analysis (NTA). Isolated RNAs were profiled with Bioanalyzer Pico kit and subjected to miRNAseq and RNAseq. EV proteins were analysed using tandem mass tag labelling. Results: The isolated EVs appeared typical at EM and were positive for the EV-marker TSG101 in WB. RNA quantity and quality proved appropriate for both miRNA and RNAseq. Different treatments affected characteristically the vesiculation from the investigated target cells of diabetes. Ninety-six EV miRNAs could characteristically discriminate between the cell types and special treatments studied. Some EV miRNAs showed treatment effects and the analysis of their target genes using KEGG disease database showed a clear link to kidney diseases. Integrated miRNA-mRNA and protein analysis was also performed. Summary/Conclusion: EV analysis provides a novel approach to reveal valuable pathophysiology, pathway and signalling information of cultured disease target cells. Changes in EV miRNAs, mRNA and proteomics may thus give valuable insight into mechanisms and targets to insulin resistance on DKD target cells. Funding: BEAt-DKD, Paulo Foundation and Novo Nordisk Foundation. PT08.05 Effects of an acute exercise on circulating extracellular vesicles: tissue-, gender- and BMI-related differences Jacopo Mariani^a, Antonello Rigamonti^a, Silvano Cella^a, Alessandra De Col^b, Federica Rota^c, Sabrina Cicolini^b, Gabriella Tringali^b, Roberta De Micheli^b, Valentina Bollati^a, Sartorio Alesandro^b and Mario Barilani ^d ^aUniversity of Milan, Department of Clinical Sciences and Community Health, Milan, Italy; ^bIstituto Auxologico Italiano, Experimental Laboratory for Auxo-endocrinological Research, Verbania and Milan, Italy; ^cEPIGET LAB, Department of Clinical Sciences and Community Health, Università degli Studi di Milano, Milan, Italy; ^dUnit of Regenerative Medicine – Cell Factory, Department of Transfusional Medicine and Hematology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano (MI), Italy; Università degli Studi di Milano, Milan, Italy Introduction: Exercise is recognized to evoke multisystemic adaptations that, particularly in obese subjects, reduce body weight, improve gluco-metabolic control, counteract sarcopenia and lower the risk of cardiometabolic diseases. Understanding the molecular mechanisms of exercise-induced benefits is of great interest due to the ensuing therapeutic implications against obesity. Aims: To characterize extracellular vesicles (EVs) in obese (F/M = 8/8; age = 21.0 ± 8.5 years, BMI = 37.9 ± 6.0 kg/m^2) and normal-weight (F/M = 4/4; age = 25.1 ± 8.2 years, BMI = 20.9 ± 1.5 kg/m^2) subjects who underwent a moderate-intensity (60% VO[2max] for 30 min or until exhaustion) exercise on a treadmill Methods: Blood samples were drawn before, at the end and during post-exercise recovery period (3 and 24 h). EVs were analysed by Nanosight and flow cytometry after labelling with the following markers: CD14+ (monocyte), CD61+ (platelet), CD62E+ (activated endothelium), CD105+ (resting endothelium), HERVW+ (human endogenous retrovirus W), SCG+ (muscle) and FABP+ (adipose tissue). Results: After exercise, 100–200 nm EVs significantly decreased (p < 0.01). There was a significantly higher post-exercise release of these EVs in normal-weight than obese subjects (p = 0.025). Considering the 30–130 nm size range, there was a significant lower release of EVs in females than males (p < 0.01). After exercise, the 130–700 nm EVs significantly decreased (p = 0.016). There was a higher release of these EVs in females than males (p = 0.05). After exercise, CD61+ EVs significantly decreased in all subjects (p = 0.02). SCG+ EVs were increased after exercise (p = 0.06). There were no significant associations of other biomarkers Summary/Conclusion: An acute exercise induces changes in the release of plasma EVs, which are tissue-, gender- and BMI-specific, suggesting that the exercise-related benefits might depend upon a complex interaction of tissue, endocrine and metabolic factors Funding: The study was supported by Progetti di Ricerca Corrente, Istituto Auxologico Italiano, IRCCS, Milan, Italy. PT08.06 Profiling of extracellular vesicles from human hepatic stellate cells Cristina Zivko ^a, Gina Valentino^a, Kathrin Fuhrmann^b, Gregor Fuhrmann^c and Paola Luciani^a ^aFriedrich Schiller University Jena, Jena, Germany; ^bHIPS, Saarbrücken, Germany; ^cHelmholtz-Institut for Pharmaceutical Research Saarland (HIPS), Saarbrücken, Germany Introduction: The role of extracellular vesicles (EVs) in intercellular communication makes them particularly interesting in the research of many pathologies, especially those diagnosed invasively. In this study we evaluated differences in the EVs shed from human hepatic stellate cells (HSCs) in their different phenotypical states, since the activation of HSCs plays a pivotal role in the progression of hepatic fibrosis, for which liver biopsy is still the diagnostic gold standard. Methods: Protocols were optimized as to induce the different activation states in the cells; untreated HSCs were compared to their activated and quiescent counterparts. EVs were isolated from conditioned cell culture medium (CCM) after differential centrifugation followed by an ultracentrifugation (UC) step, and they were purified by size exclusion chromatography (SEC). The purification was evaluated by protein content determination by bicinchoninic acid (BCA) assay. The concentration and size distribution profiles of EVs in the SEC fractions were determined by nanoparticle tracking analysis (NTA). EV-morphology was observed by scanning and cryogenic transmission electron microscopy (SEM and cryo-TEM). Results: Purification by SEC resulted in a distinct resolution between EVs and protein aggregates as determined by BCA assay. Protein content associated with EVs was only detectable in the SEC-fractions with the highest EV-yields and was comparable in all groups. Differently treated HSCs yielded EVs in similar amounts and size distributions. Quantile subtraction of the distribution curve obtained from untreated cells shows that activated HSCs produce smaller EVs (80–150 nm) more prominently than quiescent cells. SEM imaging confirmed the polydispersity in the samples. Summary/Conclusion: EVs originating from differently treated HSCs were isolated and characterized in terms of yield, size, morphology and general protein content. Our results were consistently indicative of differences in EV populations originating from the same cells in either healthy or diseased state (quiescent or activated respectively), thus creating an important basis for the potential non-invasive detection of liver diseases. Funding: The Phospholipids Research Center is gratefully acknowledged for its support to the project and Lipoid GmbH for the endowment to the University of Jena. PT08.07 Role of exosomal miR-15a in diabetic retinopathy Tengku Ain Kamalden, Anne Macgregor-Das, Nurliza Khaliddin, Nur Musfirah Mahmud, Adib Redzuan, Adil Mohamed, Hayatun Syamila Jamil, Nadia Hanib, Nur Hasyimah Azemi and Samarjit Das University of Malaya, Kuala Lumpur, Malaysia Introduction: Diabetic retinopathy is a debilitating complication of diabetes mellitus which results in irreversible blindness. Currently treatment is only initiated when significant damage from diabetic retinopathy has occurred. Early signs of damage typically remain unnoticed until it has reached advanced stages of disease. Identifying early biomarkers of disease will allow clinicians to detect the progression of disease before the onset of complications. Circulating microRNA contained in extracellular vesicles such as exosomes are potential early biomarkers and can be targeted to prevent diabetes from progressing. The aim of our project is to validate and determine the role of miR-15a as a potential early biomarker in diabetic retinopathy. Methods: This project was approved by the University of Malaya Medical Centre (UMMC) Medical Research Ethical Committee. A total of about 100 subjects (controls and patients with Type 2 DM) was recruited from UMMC, Kuala Lumpur. All subjects underwent complete eye examination and graded for diabetic retinopathy. Clinical information collected included HbA1C, renal function testing, hypertension and smoking. Extracellular vesicle (EV) isolation was performed using differential ultracentrifugation and quantified. Results: In this study, we analysed miR-15a concentrations in plasma and exosomal-enriched fractions using droplet digital and real-time PCR. There was no difference in microRNA levels in plasma observed. However, there was a significant increase in exosomal concentration (average diameter <130nM) in patients with diabetic retinopathy compared to controls (p < 0.05). There was also an increasing trend of miR-15a level among diabetic patients compared to controls. Summary/Conclusion: The findings from this study corroborated with our previous findings of increase in miR-15a levels in diabetes prior to the onset of retinopathy compared to controls. This suggests that miR-15a is involved in the early development of diabetic microvascular complications and may be a potential biomarker for early complications of diabetes. Funding: 1. Bayer Global Ophthalmology Award Program Grant. 2. University of Malaya Special Research Fund (BKS056-2017). 3. BioRad Institutional Funding (Materials and Lab consumables). 4. Fulbright Visiting Research Scholar Grant PT08.08 The effects of outer membrane vesicles delivered from Porphyromonas gingivalis on hepatic glucose metabolisms Kaya Yoshida ^a, Mariko Seyama^b, Natsumi Fujiwara^b, Hirohiko Okamura^c and Kazumi Ozaki^d ^aDepartments of Oral Healthcare Education, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan; ^bDepartments of Oral Healthcare Promotion, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan; ^cDepartment of Oral Morphology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tokushima, Japan; ^dDepartments of Oral Healthcare Promotion, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan Introduction: The outer membrane vesicles (OMVs) of Porphyromaons gingivalis (Pg), a gram-negative bacteria known as a major pathogen of periodontal diseases, include its virulence factors and regulate the aetiology of periodontal diseases by affecting microbial environment and the host cells in the oral cavity. However, it is unknown whether Pg OMVs in oral cavity could translocate to distant organ and affect the systemic diseases, whereas periodontal diseases are well known to influence the develop of diabetes mellitus. To elucidate the mechanisms by which periodontal diseases progress diabetes mellitus, we identified Pg OMV cargo proteins and verified its effects on the insulin signalling in vitro. We also analysed the translocation of Pg OMVs to the organ, and assessed the changes of hepatic glucose matabolisms in Pg OMV-treated mice. Methods: We identified the OMV cargo proteins by LC-MS/MS analyses. The effects of Pg OMV on the insulin signalling in HepG2 cells is analysed by western blot. The organ distribution of OMV was analysed by IVIS sectrum after injecting intraperitoneally Cy7-labelled Pg OMV. We also estimated the insulin sensitivity using glucose tolerance test (GTT), insulin tolerance test (ITT) in mice treated with Pg OMV for 3 weeks. Results: Pg selectively sorted its specific proteases such as arginine-specific gingipain (Rgp) and lysine-specific gingipain (Kgp) into OMVs. The treatment with Pg OMV attenuated the insulin signalling in HepG2 cells, and its effects were eliminated by OMVs from gingipain-deleated Pg. A Cy7 fluorescent signal was detected in the liver in mice injected with Cy7-labelled-Pg OMVs. The exposure of Pg OMVs for 3 weeks slightly increased casual blood glucose and insulin tolerance level in mice. Summary/Conclusion: Pg OMVs packaging gingipains were delivered to the liver, resulting in the reduction of insulin sensitivity. These capabilities of Pg OMVs may contribute to the progress of diabetes mellitus. PT09: Advances in EV Quantification and Characterization Chairs: Randy Carney; Edwin van der PolLocation: Level 3, Hall A15:30–16:30 PT09.01 Extracellular vesicle concentrations in human plasma and serum as revealed by microfluidic resistive pulse sensing and size exclusion chromatography coupled with on-line fluorescence detection Diana Kitka^a, Zoltan Varga ^b, Gergo Barta^b, Judith Mihaly^a and Jean-Luc Fraikin^c ^aRCNS HAS, Budapest, Hungary; ^bResearch Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary; ^cSpectradyne LLC, Torrance, USA Introduction: Blood is one of the most important sources of EVs in biomarker applications. However, there is a huge variation in the reported values of EV concentrations in plasma and serum in the current literature. Therefore, there is a continuous demand for new techniques for accurate determination of EV concentration. The aim of this study was to characterize EVs in normal plasma and serum using novel techniques such as microfluidic resistive pulse sensing (MRPS) and size exclusion chromatography (SEC) coupled with on-line fluorescence detection. Methods: To obtain cell free serum and plasma, blood was collected from healthy volunteers using serum activator and EDTA vacutainer tubes, respectively. Cells were removed by centrifugation at 2500 x g twice. Samples were further purified with a Sepharose CL-2B gravity column and analysed by MRPS using the nCS1 instrument (Spectradyne LLC, USA). For the fluorescence SEC experiments, samples were labelled with PE-conjugated anti-CD61 and analysed with a JASCO (Japan) liquid chromatography system supplemented with an FP-2020 fluorescence detector and using a 1 mL column filled with CL-2B gel. Results: The particle concentrations of serum and plasma determined by MRPS in the 65–250 nm size range were 2.06E+10 1/mL and 1.77E+10 1/mL, respectively. In the 250–2000 nm range, we found 2.22E+8 1/mL and 5.50E+7 1/mL for serum and plasma. These concentrations correspond to 0.29 E+10 1/mL increase for the smaller size range, and 1.67E+8 1/mL for the larger size range, which can be accounted for the EVs produced during clotting. Fluorescence SEC experiments with PE-CD61 revealed that the percentage of CD61 bound to EVs increased from 2.25% (plasma) to 36% (serum). Using these data, we obtained that one platelet-derived EV contains approx. 15 CD61 glycoproteins in average. Summary/Conclusion: By the combination of MRPS and fluorescence SEC we quantified the overall particle concentrations in serum and plasma, and using a platelet-specific fluorescently labelled antibody, we determined the average number of CD61 glycoproteins on platelet-derived EVs formed during blood clotting. Funding: This work was supported under grant numbers PD 121326 and NVKP_16-1-2016-0007 by NKFIH (Hungary). ZV was supported by the János Bolyai Research Fellowship. PT09.02 The nanobioanalytical platform, a tuneable tool for a sensitive detection & characterization of extracellular vesicles subsets from biological samples Balasubramaniam Namasivayam^a, Yu-Wen Wu^b, Liling Delila^b, Annie Frelet-Barrand^a, Thierry Burnouf^b, Celine Elie-Caille^a and Wilfrid Boireau ^a ^aFEMTO-ST Institute, UBFC, CNRS, Besançon, France; ^bCollege of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan (Republic of China) Introduction: The NanoBioAnalytical (NBA) platform is an established, calibrated and label-free system to characterize Extracellular Vesicles (EVs), without limitation in size, in different biological samples [1, 2]. NBA benefits were recently highlighted in latest MISEV guidelines [3]. The NBA platform combines biodetection and phenotyping of EVs subsets by immunocapture monitored by Surface Plasmon Resonance (SPR) on biochip, followed by EVs quantitation and sizing thanks to metrological evaluation by Atomic Force Microscopy (AFM). Our aim is to push the limit of the NBA to address clinical studies involving EVs. Methods: We emphasise here the performance of the NBA platform for establishing its dynamic range and limit of detection (LOD) for blood derived EVs. Concentration of EVs was first determined in solution by Tunable Resistive Pulse Sensing; NBA sensitivity and reliability was then studied by SPR on biochips presenting a-CD41 antibody arrays. Finally, even on 1000-fold diluted samples, reliable and complementary information to SPR measurements on size distribution, counting and shape deciphering could be obtained by AFM. Results: Optimizing different factors (flow rate, density of receptors on the surface, etc.) enabled detection of blood derived EVs at dynamic range from 10^6 to 10^9 particles /mL on a-CD41 surface. The determination of the LOD of EVs and their subsets size distribution at different capture levels are currently in progress. Summary/Conclusion: The NBA platform is modular and capable of detecting EVs reliably even in highly diluted samples. Such characterization and correlation studies are crucial for accurate and comprehensive characterization of EVs in biological samples with good reproducibility. References